Metastatic Colon Cancer Cells Research Articles

Evaluating the Anti-proliferative and Apoptotic Role of Atrial Natriuretic Peptide in Colon Cancer Cell Lines

The use of small peptides and conventional anticancer drugs is gaining importance in oncology as small peptides may help increase chemo or radiation sensitivity. The present study aimed to study the impact of atrial natriuretic peptide (ANP) in reducing colon cancer cell proliferation in primary and metastatic colon cancer cell lines. The proliferation of colon cancer cell lines (SW480 and SW620) was analysed by CCK-8 assay and cell damage was analyzed by lactate dehydrogenase activity. Catalase activity assay was performed to measure the oxidative stress and antioxidant defense mechanisms in SW480 and SW620 cell lines. Subsequently, up or downregulation of cancer-specific gene expression such as BAX, Caspase-3, BCL-2, CDK-6, and PCNA genes were assessed after the treatment of small peptide ANP in SW480 and SW620 colon cancer cell lines. The ANP treatment decreased the colon cancer cell proliferation, upregulated the apoptosis-related gene expression (Caspase-3, BAX), downregulated the anti-apoptotic gene (BCL-2) expression, and proliferation-related genes (CDK-6, PCNA) in SW480 and SW620 colon cancer cell lines, and the differences were found to be statistically significantly. Further, increased levels of catalase in the colon cancer cell lines after ANP-treatment suggested the therapeutic role of ANP. Subsequently, the LDH analysis showed the potential of ANP in inducing colon cancer cell damage. Collectively, the current study clearly shows that ANP is a potential molecule in reducing uncontrolled cancer cell growth. However, additional research using animal models and additional colon cancer cell lines is needed to validate its potential usage in clinical studies.

Abstract 447: Anti-tumor efficacy of ompenaclid, a creatine transporter inhibitor, in KRAS mutant tumors, including NSCLC

Abstract Background: Pre-clinical studies identified creatine metabolism to be activated in KRAS mutant metastatic colorectal cancer (CRC) as a mechanism to fuel the increased energy demands of RAS mutant tumors. Metastatic colon cancer cells overexpress and subsequently release creatine kinase-B (CKB) into the extracellular space, where it phosphorylates creatine to generate the high energy metabolite phosphocreatine. Ompenaclid is a small molecule inhibitor of the creatine transporter SLC6A8. Inhibition of SLC6A8 by ompenaclid prevents import of phosphocreatine/creatine, resulting in depletion of intracellular creatine, phosphocreatine and ATP levels. This results in tumor apoptosis and robust anti-tumor efficacy in CRC models with a wide range of KRAS mutant alleles. Ompenaclid is currently in a Phase 2 trial, in combination with FOLFIRI and bevacizumab, in patients with RAS-mutated second-line advanced/metastatic colorectal cancer. Here, we assessed efficacy of ompenaclid in tumors beyond CRC, including KRAS WT and KRAS mutant NSCLC. Methods: Athymic mice were inoculated with CRC, pancreatic, gastric or NSCLC cancer cell lines and treated with ompenaclid once tumors reached ~150 mm3, until termination. Plasma metabolites were quantified by LC-MS/MS and tumoral expression of CKB and SLC6A8 in NSCLC and CRC tumors was measured by qPCR after treatment with a control or ompenaclid-supplemented diet for ~10 days. Combination treatment with MRTX849, a KRAS G12C inhibitor, was conducted in a pancreatic MiaPaca xenograft model. Results: Our studies demonstrate ompenaclid anti-tumor efficacy in NSCLC and pancreatic xenografts. Notably, and consistent with findings in CRC, efficacy in NSCLC was more robust in RAS mutant cancer models, relative to those bearing KRAS WT tumors. Metabolite analysis of the plasma of tumor-bearing mice at baseline revealed that mice harboring RAS mutant tumors had higher levels of p-creatine and lower levels of ATP, indicating that RAS mutant tumors utilize higher amounts of ATP, which may be converted to phosphocreatine by tumoral CKB, as confirmed by qPCR analysis. This finding is consistent with metastatic RAS mutant CRC tumors utilizing extracellular ATP to generate phosphocreatine and thus exhibiting greater dependency on creatine metabolism relative to RAS WT tumors. Treatment with ompenaclid also demonstrated additive efficacy in combination with KRAS G12C inhibitors. In summary, these data support development of ompenaclid in indications beyond CRC, including NSCLC, as well as in combination with KRAS G12C inhibitors. Citation Format: PuiChi Lo, Victor Sanz Chavez, Shugaku Takeda, Masoud Tavazoie, Isabel Kurth. Anti-tumor efficacy of ompenaclid, a creatine transporter inhibitor, in KRAS mutant tumors, including NSCLC [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 447.

Anti-Cancer Mechanisms of Diarylpentanoid MS17 (1,5-Bis(2-hydroxyphenyl)-1,4-pentadiene-3-one) in Human Colon Cancer Cells: A Proteomics Approach.

Diarylpentanoids are synthesized to overcome curcumin's poor bioavailability and low stability to show enhanced anti-cancer effects. Little is known about the anti-cancer effects of diarylpentanoid MS17 (1,5-bis(2-hydroxyphenyl)-1,4-pentadiene-3-one) in colon cancer cells. This study aimed to elucidate molecular mechanisms and pathways modulated by MS17 in colon cancer based on proteomic profiling of primary SW480 and metastatic SW620 colon cancer cells. Cytotoxicity and apoptotic effects of MS17 were investigated using MTT assay, morphological studies, and Simple Western analysis. Proteomic profiling using LC/MS analysis identified differentially expressed proteins (DEPs) in MS17-treated cells, with further analysis in protein classification, gene ontology enrichment, protein-protein interaction network and Reactome pathway analysis. MS17 had lower EC50 values (SW480: 4.10 µM; SW620: 2.50 µM) than curcumin (SW480: 17.50 µM; SW620: 13.10 µM) with a greater anti-proliferative effect. MS17 treatment of 1× EC50 induced apoptotic changes in the morphology of SW480 and SW620 cells upon 24 h treatment. A total of 24 and 92 DEPs (fold change ≥ 1.50) were identified in SW480 and SW620 cells, respectively, upon MS17 treatment of 2× EC50 for 24 h. Pathway analysis showed that MS17 may induce its anti-cancer effects in both cells via selected DEPs associated with the top enriched molecular pathways. RPL and RPS ribosomal proteins, heat shock proteins (HSPs) and ubiquitin-protein ligases (UBB and UBC) were significantly associated with cellular responses to stress in SW480 and SW620 cells. Our findings suggest that MS17 may facilitate the anti-proliferative and apoptotic activities in primary (SW480) and metastatic (SW620) human colon cancer cells via the cellular responses to stress pathway. Further investigation is essential to determine the alternative apoptotic mechanisms of MS17 that are independent of caspase-3 activity and Bcl-2 protein expression in these cells. MS17 could be a potential anti-cancer agent in primary and metastatic colon cancer cells.

Mechanism of bufalin inhibition of colon cancer liver metastasis by regulating M2-type polarization of Kupffer cells induced by highly metastatic colon cancer cells.

Patients with metastatic colorectal cancer often have poor outcomes, primarily due to hepatic metastasis. Colorectal cancer (CRC) cells have the ability to secrete cytokines and other molecules that can remodel the tumor microenvironment, facilitating the spread of cancer to the liver. Kupffer cells (KCs), which are macrophages in the liver, can be polarized to M2 type, thereby promoting the expression of adhesion molecules that aid in tumor metastasis. Our research has shown that huachanshu (with bufalin as the main active monomer) can effectively inhibit CRC metastasis. However, the underlying mechanism still needs to be thoroughly investigated. We have observed that highly metastatic CRC cells have a greater ability to induce M2-type polarization of Kupffer cells, leading to enhanced metastasis. Interestingly, we have found that inhibiting the expression of IL-6, which is highly expressed in the serum, can reverse this phenomenon. Notably, bufalin has been shown to attenuate the M2-type polarization of Kupffer cells induced by highly metastatic Colorectal cancer (mCRC) cells and down-regulate IL-6 expression, ultimately inhibiting tumor metastasis. In this project, our aim is to study how high mCRC cells induce M2-type polarization and how bufalin, via the SRC-3/IL-6 pathway, can inhibit CRC metastasis. This research will provide a theoretical foundation for understanding the anti-CRC effect of bufalin.

Exosomes Derived from Metastatic Colon Cancer Cells Induced Oncogenic Transformation and Migratory Potential of Immortalized Human Cells

Tiny vesicles, synthesized from cell membranes through endocytosis, form multivesicular bodies (MVBs). These MVBs can either undergo lysosomal degradation or be released to the extracellular environment. The vesicle that ranges in size from 30-300 nm that were positive for markers such as CD63 or CD9 are known as exosomes. Exosomes are shown to contain various biological macromolecules such as DNA, RNA, miRNA, proteins, and lipids. Releasing exosomes from cells to the extracellular environment is an important strategy to transfer information between the cells. Exosomes from primary colon cancer cells have been shown to induce the oncogenic transformation of normal cells. In this study, we have demonstrated that treating normal cells (HEK) with exosomes derived from the metastatic colon cancer cell line (SW620) increases cell proliferation, allowing the normal cells to grow anchored independently, suggesting the oncogenic transformation of normal cells. In addition, there was an increase in migration and invasion properties of HEK cells exposed to exosomes from SW620 cells. Our results suggest that following the treatment with metastatic colon cancer cell-derived exosomes, the HEK cells underwent an oncogenic transformation and Epithelial-Mesenchymal Transition (EMT) that will allow the cancerous cells to metastasize.

Quercetin Change the Exosome Secretion and Total miRNA Concentration in Primary (Colo320) and Metastatic (Colo741) Colon Cancer Cell Lines

Quercetin Change the Exosome Secretion and Total miRNA Concentration in Primary (Colo320) and Metastatic (Colo741) Colon Cancer Cell Lines

BMAP-27 Peptide Reduces Proliferation and Increases Apoptosis in Primary and Metastatic Colon Cancer Cell Lines

BMAP-27 peptide is reported to possess apoptotic and anti-proliferative effects against cancer cells but the actual mechanism of action is yet to be investigated. In the current investigation, we aimed to study the role of the BMAP-27 peptide in reducing proliferation and increasing apoptosis in colon cancer cell lines. In this study, we used primary and metastatic colon cancer cell lines SW480 and SW620. Cell proliferation was measured using MTT and CCK-8 assays, and cellular damage was analyzed by lactate dehydrogenase assay. Apoptosis, cell cycle, and proliferation potentials were measured by the expression of CASPASE3, BAX, BCL-2, TP53, CDK-6, PCNA, WNT11, AXIN1, and CTNNB1 genes. Additionally, in-silico studies were conducted to determine the binding affinities of BMAP-27 with adenomatous polyposis coli (APC) and β-catenin proteins, one of the primary regulators of colon cancer. BMAP-27 peptide reduced colon cancer cell proliferation, upregulated tumor suppressor genes CASPASE3, BAX, TP53, AXIN1 expression, and downregulated the expression of oncogenes BCL-2, CDK-6, PCNA, WNT11, CTNNB1 in both the cell lines, however, in the primary colon cancer cell line the changes are found to be more significant. The molecular dynamic simulation analysis revealed substantial binding affinity of the peptide to APC and β-catenin proteins. BMAP-27 peptide significantly inhibited the proliferation and induced apoptosis in the primary colon cancer cell line than in the metastatic colon cancer cell line. In-silico results suggest that BMAP-27 shows a strong binding affinity with APC and β-catenin proteins, highlighting its role in inhibiting colon cancer cell proliferation.

Exosomes from metastatic colon cancer cells drive the proliferation and migration of primary colon cancer through increased expression of cancer stem cell markers CD133 and DCLK1

The exchange of biological material between the neighbouring cells is essential for homeostasis. In pathological conditions, such as cancer, the major challenge in cancer treatment is the abnormal expression of crucial proteins and miRNA exchanged between the cancer cells through extracellular vesicles called exosomes. Clinically, it has been noticed that the primary tumour and the distal metastases are interconnected and co-dependent. The exosomes are key factors responsible for preparing the pre-metastatic niche and communicating between the tumour and the distal metastatic site. Cancer stem cells (CSCs) are a subpopulation of cancer cells with self-renewal characteristics and are shown to be responsible for metastasis. This study aims to understand the effect of metastatic cell line-derived exosomes and their regulation of CSC marker expressions on primary colon cancer cell lines. We have identified that treatment of primary colon cancer cell lines with metastatic colon cancer cell-derived exosomes has significantly increased the proliferation, colony formation, cell migration, and invasion. In addition, there was a significant increase in the number and size of spheroids following the exosomes treatment. We found that this metastatic phenotype is due to the increased expression of CD133 and DCLK1 in primary colon cancer cells.

Ceramide Analog 5cc Overcomes TRAIL Resistance by Enhancing JNK Activation and Repressing XIAP Expression in Metastatic Colon Cancer Cells

Introduction: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered to be an effective apoptosis inducer due to its selectivity for tumor cells. However, many cancer cells, especially metastatic cancer cells, often exhibit resistance to TRAIL because their apoptotic pathway is impaired or their pro-survival pathway is overactivated. TRAIL resistance is the main obstacle to current TRAIL therapy. Nowadays, ceramide analogs represent a new class of potential anticancer agents. Therefore, we hypothesized that disrupting pro-survival signaling with ceramide analogs would increase TRAIL-mediated apoptosis. Methods: MTT assay and flow cytometry were conducted to evaluate the synergistic effect of ceramide analog 5cc on TRAIL in metastatic colon cancer cells. Western blot was used to detect signaling proteins affected by 5cc. RNA interference was performed to analyze the effects of specific gene on 5cc-enhanced apoptosis. Results: Ceramide analog 5cc markedly enhanced TRAIL-induced apoptosis evidenced by increased propidium iodide/annexin V double-positive cells and PARP cleavage in SW620 and LS411N cells. At the molecular level, 5cc significantly reduced the expression of anti-apoptotic protein X-linked inhibitor of apoptosis protein (XIAP) through the activation of the c-Jun n-terminal kinase (JNK) pathway which is critically involved in sensitizing tumor cells to TRAIL/5cc combination. JNK-silenced cells exhibited a significant reversal of TRAIL/5cc-mediated apoptosis. Conclusion: Our data demonstrated that ceramide analog 5cc overcomes TRAIL resistance by enhancing JNK activation and repressing XIAP expression in metastatic colon cancer cells.

Artificial Diets with Altered Levels of Sulfur Amino Acids Induce Anticancer Activity in Mice with Metastatic Colon Cancer, Ovarian Cancer and Renal Cell Carcinoma.

Sulfur-containing amino acids methionine (Met), cysteine (Cys) and taurine (Tau) are common dietary constituents with important cellular roles. Met restriction is already known to exert in vivo anticancer activity. However, since Met is a precursor of Cys and Cys produces Tau, the role of Cys and Tau in the anticancer activity of Met-restricted diets is poorly understood. In this work, we screened the in vivo anticancer activity of several Met-deficient artificial diets supplemented with Cys, Tau or both. Diet B1 (6% casein, 2.5% leucine, 0.2% Cys and 1% lipids) and diet B2B (6% casein, 5% glutamine, 2.5% leucine, 0.2% Tau and 1% lipids) showed the highest activity and were selected for further studies. Both diets induced marked anticancer activity in two animal models of metastatic colon cancer, which were established by injecting CT26.WT murine colon cancer cells in the tail vein or peritoneum of immunocompetent BALB/cAnNRj mice. Diets B1 and B2B also increased survival of mice with disseminated ovarian cancer (intraperitoneal ID8 Tp53-/- cells in C57BL/6JRj mice) and renal cell carcinoma (intraperitoneal Renca cells in BALB/cAnNRj mice). The high activity of diet B1 in mice with metastatic colon cancer may be useful in colon cancer therapy.

Molecular Characterization of Primary and Metastatic Colon Cancer Cells to Identify Therapeutic Targets with Natural Compounds.

Metastasis is the world's leading cause of colon cancer morbidity. Due to its heterogeneity, it has been challenging to understand primary to metastatic colon cancer progression and find a molecular target for colon cancer treatment. The current investigation aimed to characterize the immune and genotypic profiles of primary and metastatic colon cancer cell lines and identify a molecular target for colon cancer treatment. Colony-forming potential, migration and invasion potential, cytokine profiling, miRNA, and mRNA expression were examined. Molecular docking for the Wnt signaling proteins with various plant compounds was performed. Colony formation, migration, and invasion potential were significantly higher in metastatic cells. The primary and metastatic cells' local immune and genetic status revealed TGF β-1, IL-8, MIP-1b, I-TAC, GM-CSF, and MCP-1 were highly expressed in all cancer cells. RANTES, IL-4, IL- 6, IFNγ, and G-CSF were less expressed in cancer cell lines. mRNA expression analysis displayed significant overexpression of proliferation, cell cycle, and oncogenes, whereas apoptosis cascade and tumor suppressor genes were significantly down-regulated in metastatic cells more evidently. Most importantly, the results of molecular docking with dysregulated Wnt signaling proteins shows that peptide AGAP and coronaridine had maximum hydrogen bonds to β-catenin and GSK3β with a better binding affinity. This study emphasized genotypic differences between the primary and metastatic colon cancer cells, delineating the intricate mechanisms to understand the primary to metastatic advancement. The molecular docking aided in understanding the future molecular targets for bioactive- based colon cancer therapeutic interventions.

Two circPPFIA1s negatively regulate liver metastasis of colon cancer via miR-155-5p/CDX1 and HuR/RAB36

BackgroundCircular RNAs (circRNAs) play a critical role in colorectal cancer (CRC) progression, including metastasis. However, the detailed molecular mechanism is not fully understood.MethodsDifferentially expressed circRNAs between primary KM12C and liver metastatic KM12L4 colon cancer cells were identified by microarray. The expression of circRNAs was measured by semi-quantitative (semi-qPCR) and real time-quantitative PCR (RT-qPCR). Metastatic potential including invasive and migratory abilities, and liver metastasis were examined by transwell assays and intrasplenic injection, respectively. CircPPFIA1-associated microRNA (miRNA) and RNA-binding protein (RBP) were screened by an antisense oligonucleotide (ASO) pulldown experiment. The effects of circPPFIA1 on target gene expression were evaluated by RT-qPCR and western blot analyses.ResultsBy analyzing circRNA microarray data, we identified two anti-metastatic circRNAs generated from PPFIA1 with different length, which named circPPFIA1-L (long) and -S (short). They were significantly downregulated in liver metastatic KM12L4 cells compared to primary KM12C cells. The knockdown of circPPFIA1s in KM12C enhanced metastatic potential and increased liver metastasis. Conversely, overexpression of circPPFIA1s weakened metastatic potential and inhibited liver metastasis. circPPFIA1s were found to function as sponges of oncogenic miR-155-5p and Hu antigen R (HuR) by an ASO pulldown experiment. circPPFIA1s upregulated tumor-suppressing CDX1 expression and conversely downregulated oncogenic RAB36 by decoying miR-155-5p and by sequestering HuR, respectively.ConclusionOur findings demonstrate that circPPFIA1s inhibit the liver metastasis of CRC via the miR-155-5p/CDX1 and HuR/RAB36 pathways.

Sulforaphane has an additive anticancer effect to FOLFOX in highly metastatic human colon carcinoma cells.

Colorectal cancer (CRC) is one of the most common malignancies worldwide. Patients with CRC may need chemotherapy (CTx) in a neoadjuvant, adjuvant or palliative setting through the course of the disease. Unfortunately, its effect is limited by chemoresistance and chemotoxicity. Novel more effective and non‑toxic CTx regimens are needed to further improve CRC treatment outcomes. Thus, the present study was designed to test the hypothesis that non‑toxic sulforaphane (SF) is effective against CRC and has additive effects in combination with conventional 5‑fluorouracil, oxaliplatin and folinic acid (FOLFOX) CTx invitro. Highly metastatic human colon cancer cells, CX‑1, and fibroblasts were treated with FOLFOX ± SF. Cell viability was assessed using an MTT assay. The level of apoptosis and the expression of apoptotic proteins were measured by TUNEL assay and quantitative PCR analysis. Aldehyde dehydrogenase isoform 1 (ALDH1) and multidrug resistance protein 2 (MRP2) levels were evaluated. The ability of cells to form spheroids was measured in three‑dimensional cell culture. SF alone and in combination with FOLFOX effectively decreased the viability of the CX‑1 cells, promoted apoptosis within the CX‑1 cells, prevented cellular spheroid formation and decreased ALDH1 activity. However, SF promoted MRP2 expression and protein levels. In conclusion, SF together with conventional FOLFOX has additive anticancer effects against highly metastatic human CRC invitro.

Extracellular vesicle expansion of PMIS-miR-210 expression inhibits colorectal tumour growth via apoptosis and an XIST/NME1 regulatory mechanism.

BackgroundColorectal cancer (CRC) has a high mortality rate, and therapeutic approaches to treat these cancers are varied and depend on the metabolic state of the tumour. Profiles of CRC tumours have identified several biomarkers, including microRNAs. microRNA‐210 (miR‐210) levels are directly correlated with CRC survival. miR‐210 expression is higher in metastatic colon cancer cells versus non‐metastatic and normal colon epithelium. Therefore, efficient methods to inhibit miR‐210 expression in CRC may provide new advances in treatments.MethodsExpression of miRs was determined in several metastatic and non‐metastatic cell lines. miR‐210 expression was inhibited using PMIS‐miR‐210 in transduced cells, which were transplanted into xenograft mice. In separate experiments, CRC tumours were allowed to grow in xenograft mice and treated with therapeutic injections of PMIS‐miR‐210. Molecular and biochemical experiments identified several new pathways targeted by miR‐210 inhibition.Results miR‐210 inhibition can significantly reduce tumour growth of implanted colon cancer cells in xenograft mouse models. The direct administration of PMIS‐miR‐210 to existing tumours can inhibit tumour growth in both NSG and Foxn1nu/j mouse models and is more efficacious than capecitabine treatments. Tumour cells further transfer the PMIS‐miR‐210 inhibitor to neighbouring cells by extracellular vesicles to inhibit miR‐210 throughout the tumour. miR‐210 inhibition activates the cleaved caspase 3 apoptotic pathway to reduce tumour formation. We demonstrate that the long non‐coding transcript XIST is regulated by miR‐210 correlating with decreased XIST expression in CRC tumours. XIST acts as a competing endogenous RNA for miR‐210, which reduces XIST levels and miR‐210 inhibition increases XIST transcripts in the nucleus and cytoplasm. The increased expression of NME1 is associated with H3K4me3 and H3K27ac modifications in the NME1 proximal promoter by XIST.ConclusionDirect application of the PMIS‐miR‐210 inhibitor to growing tumours may be an effective colorectal cancer therapeutic.

Is Acteoside Effects on Colon Cancer Stem Cells Via Inflamation or Apoptosis?

AIM: Colon cancer is one of the main health problems worldwide. Cancer stem cells (CSCs) are referred to as tumor-initiating cells involved in tumor heterogeneity and dormancy. CSCs can cause drug resistance, metastasis, and recurrence of primary and metastatic cancers. The interactions and survival trends of colon cancer stem cells with other cells may be an alternative route for effective treatment. In our study, we aimed to evaluate the effects of asteoside on stem cell properties, apoptotic and inflammatory processes in primary (HCT-116) and metastatic (Colo-741) colon CSCs. METHERIALS AND METHODS: CSCs were obtained from both types of colon cancer cell lines with the MINIMACS system using the anti-CD133 reagent. Metastatic Colo-741 and non-metastatic HCT-116 CD133+ and CD133- cells were cultured with or without Acteoside for 48 hours. Expressions of Caspase-3, Bcl, Bax, and Fas-L for apoptosis, and IL-1β, TNF-α, IL-6, IL8 and IL-10 for inflammation were analyzed by indirect immunocytochemistry technique by performing H-Score. Changes in cell morphology were examined under an inverted microscope. RESULTS AND CONCLUSION: It was observed that Oct-4 expression decreased after acteocyte administration in both metastatic and non-metastatic colon cancer cells. In addition, Colo-741 increased the intensity of the Bax/Bcl ratio in both CD133+ and CD133- cells and increased caspase 3 expression. While acteoside did not immunohistochemically affect inflammation in metastatic COLO-741 cells, it contributed to the apoptotic process by changing the Bax/Bcl ratio. When the morphology of the cells was evaluated, it was observed that the number of apoptotic cells increased in COLO-741 CD133+ and CD133- cells.

Exploring dithiolate-amine binary ligand systems for the supramolecular assemblies of Ni(II) coordination compounds: Crystal structures, theoretical studies, cytotoxicity studies, and molecular docking studies

Dithiolate-amine binary ligand systems based two novel supramolecular structures of Ni(II) coordination compounds [Ni(2-ap)2(i-mnt)] (1) and [Ni2(i-mnt)2(tn)2]n (2) [where i-mnt−2: 1,1-dicyanoethylene-2,2-dithiolate, 2-ap: 2-amino pyridine, tn: 1,3-diaminopropane] have been designed, synthesized and characterized by spectroscopic techniques and X-ray crystallography. In complex 1, the N2S2 coordination kernel around Ni+2 attains distorted square planar geometry (Okuniewski parameter τ/4 = 0.17) and its supramolecular array has a dominant influence of N H···N type hydrogen bonds forming significant ring geometry of graph-set-motifR428. The i-mnt−2 adopts a diverse coordination mode in 2 contrary to 1 resulting in a binuclear one-dimensional infinite polymeric chain structure of consecutive NiS4 [square planar, S4 donor set τ/4 = 0] and NiN6 (octahedral) coordination kernels linked by i-mnt−2 that runs along [1 0 0] axis wherein N H···S, N H···N, C H···S type hydrogen bonds are believed to be crystal structure stabilizers. The Hirshfeld surface analyses (HS) at the molecular and atomic levels have been extensively studied to quantify all non-covalent interactions/contacts and the nature of ligand - metal interactions. The prominent N···H/H···N [28.1 % (1), 27.3 % (2A, NiS4), 20 % (2B, NiN6)] and S···H/H···S contacts [10.4 % (1), 32.8 % (2A), 29 % (2B)] presumably play vital role in crystal packing. The non-covalent interactions were further investigated by Molecular Electrostatic Potential (MEP) surface at PBE0-D3/def2-TZVP level and QTAIM optimizations (for 1). The MTT assay study using SW620 metastatic colon cancer cells shows a significant dose-dependent decline in cell proliferation with increasing concentration for 1 but for 2, the reverse trend is followed. A molecular docking study with the APC protein has further investigated the antiproliferative activity of 1 (MTT assay).

Abstract 5632: Combinatorial epigenetic targeting of PRC2 complexes upregulates MHC antigen presentation components in melanoma and gastrointestinal cancer cells

Abstract Low tumor immunogenicity is associated with immune escape, immunotherapy resistance, and poor patient survival [1-3]. Major histocompatibility complex (MHC) antigen presentation pathways (APPs) play a crucial role in promoting tumor T-cell recognition and initiating antitumoral immune responses. However, epigenetic silencing of APPs is a common mechanism in various cancers, including melanoma and gastrointestinal malignancies [3,4]. In the current investigation, mouse melanoma (B16/OVA) and human colon cancer (LOVO and HCT116), small intestine cancer (HUTU80), and normal colonic epithelial (CCD841) cell lines were treated with epigenetic drug candidates. As reported [5], a decrease in histone H3K27 methylation marks via polycomb repressive complex 2 (PRC2) inhibitors, as well as altered histone acetylation status from histone deacetylase 3 (HDAC3)-specific inhibition, increased the expression of APP components, based on immunoblotting and RT-qPCR experiments. Normal colonic epithelial cells showed resistance to the treatments, indicating cancer cell-specific effects of the test agents. In B16/OVA cells, flow cytometry and enzyme linked immunosorbent assays corroborated that HDAC3 and/or PRC2 inhibitor combinations increased cell surface occupancy of MHC-I complexes and increased CD8+ T-cell activation. These findings have implications for the clinical application of next-generation combinatorial epigenetic agents [6,7], targeted towards increased tumor immunogenicity and enhanced efficacy of new immunoepigenetic strategies. Supported in part by NCI grant CA122959, by the John S. Dunn Foundation, and by a Chancellor’s Research Initiative.

Abstract 5713: Alternative splicing of the chromatin modifier gene BAZ1A in colorectal cancer

Abstract Bromodomain Adjacent to Zinc finger Domain 1A (BAZ1A) is a non-catalytic subunit of the ATP-dependent chromatin assembly factor that regulates key cellular processes such as DNA replication, chromatin assembly, DNA repair, and transcriptional gene regulation (1). Defective DNA repair is a hallmark of cancer etiology, and BAZ1A is one of the deregulated proteins in colorectal cancer. The BAZ1A transcript exhibits full-length and truncated forms, generated via alternative splicing of exon-13. We observed that the histone deacetylase inhibitor sulforaphane (SFN) modulated alternative splicing of BAZ1A in human colon cancer cells by increasing the relative expression of the short vs. the long transcript. Cell Cycle and Apoptosis Regulator 2 (CCAR2) and Zinc finger protein 326 (ZIRD) are components of the DBIRD complex that integrates alternative splicing and the rate of transcript elongation (2). Knockdown of DBIRD members, including ZIRD and splicing factor hnRNPA1, downregulated expression of BAZ1A long form, whereas the BAZ1A short form was unaltered. Previous studies in SFN-treated colon cancer cells revealed that the BAZ1A bromodomain recognized a novel K97 acetylation site in CCAR2 (3). Co-immunoprecipitation analyses revealed that CCAR2 and BAZ1A interacted with each other in the presence and absence of SFN. On investigating the involvement of CCAR2 in BAZ1A alternative splicing, SFN treatment increased the expression of both BAZ1A long and short forms in CCAR2-/- HCT116 colon cancer cells. Although the total expression of BAZ1A was influenced by CCAR2 loss, alternative splicing of BAZ1A was independent of DBIRD complex-mediated regulation. Pursuing the role of p53 and p21 in BAZ1A alternative splicing, SFN treatment increased the expression of BAZ1A short form in p21-/- HCT116 cells, whereas this was impaired in p53-/- HCT116 cells. Overall, this investigation provided mechanistic insights into the regulation of alternative splicing in SFN-treated colon cancer cells, and BAZ1A functionalities that might be targeted via next-generation combinatorial epigenetic agents (4). Supported in part by NCI grant CA122959, by the John S. Dunn Foundation, and by a Chancellor’s Research Initiative.

Melatonin and andrographolide synergize to inhibit the colospheroid phenotype by targeting Wnt/beta-catenin signaling.

β-catenin signaling, and angiogenesis are associated with colospheroid (CSC), development. CSCs, spheroids derived from colon cancer cells, are responsible for metastasis, drug resistance, and disease recurrence. Whether dysregulating β-catenin and inhibiting angiogenesis reduce CSC growth is unknown. In this study, the molecular mechanism of CSC growth inhibition was evaluated using a novel combination of melatonin (MLT) and andrographolide (AGP). These drugs have anticarcinogenic, antioxidant, and antimetastatic properties. CSCs were obtained from two metastatic colon cancer cell lines (HT29 and HCT-15). The viability and stemness were monitored (FDA propidium iodide staining and immunoblot for CD44, CD133, Nanog, Sox2, and Oct4). The drug combination synergistically diminished stemness via increased reactive oxygen species (ROS) levels, reduced mitochondrial membrane potential and ATP level. MLT + AGP induced cell death by inhibiting β-catenin expression and its downregulatory signals, Cyclin D1, c-Myc. MLT + AGP treated cells exhibited translocation of phospho-β-catenin to the nucleus and dephosphorylated-β-catenin. Downregulation of β-catenin activation and its transcription factors (TCF4 and LEF1) and GTP binding/G-protein related activity were found in the dual therapy. Angiogenic inhibition is consistent with downregulation of VEGF messenger RNA transcripts (VEGF189), phosphorylated VEGF receptor protein expression, matrigel invasion, and capillary tube inhibition. In vivo, the intravenous injection of MLT + AGP slowed HT29 metastatic colon cancer. Histopathology indicated significant reduction in microvascular density and tumor index. Immunohistochemistry for caspase 7, and β-catenin found increased apoptosis and downregulation of β-catenin signals. The mechanism(s) of decreased colospheroids growth were the inhibition of the Wnt/β-catenin pathway. Our results provide a rationale for using MLT in combination with AGP for the inhibition of CRCs.

Celastrol and Resveratrol Modulate SIRT Genes Expression and Exert Anticancer Activity in Colon Cancer Cells and Cancer Stem-like Cells.

Simple SummaryThe recovery rate in patients with metastatic colorectal cancer (CRC) remains low and declines with successive lines of treatment. This phenomenon is caused by the development of drug resistance and the presence of colorectal cancer stem cells (CSCs). Phytochemicals, like -celastrol and resveratrol, are very promising for colon cancer therapy, owing to their low or no toxicity and their pleiotropic activity, enabling them to interact with various biological targets. In the present study, the potential anticancer mechanisms of both compounds against metastatic colon cancer cells and the capacity to eradicate CSCs were investigated.Metastatic colorectal cancer (CRC) remains a hard-to-cure neoplasm worldwide. Its curability declines with successive lines of treatment due to the development of various cancer resistance mechanisms and the presence of colorectal cancer stem cells (CSCs). Celastrol and resveratrol are very promising phytochemicals for colon cancer therapy, owing to their pleiotropic activity that enables them to interact with various biological targets. In the present study, the anticancer activities of both compounds were investigated in metastatic colon cancer cells (LoVo cells) and cancer stem-like cells (LoVo/DX). We showed that celastrol is a very potent anti-tumor compound against metastatic colon cancer, capable of attenuating CSC-like cells at the molecular and cellular levels. In contrast, resveratrol has a much greater effect on colon cancer cells that are expressing standard sensitivity to anticancer drugs, than on CSC-like cells. In addition, both polyphenols have different influences on the expression of SIRT genes, which seems to be at least partly related to their anti-tumor activity.