Colo205 Cells Research Articles

In vitro anti-inflammatory and comparative cytotoxicity studies on methanolic extract of Enicostemma hyssopifolium

Enicostemma hyssopifolium is a perineal herb widely used as an antidiabetic agent in folklore medicine. The present work was focused on the assessment of its anti-inflammatory and cytotoxic properties. Its methanolic extract was evaluated for anti-inflammatory activity using murine monocytic macrophage RAW 264.7 cells, and screened for its cytotoxic property in different cancer cell lines. The methanolic extract was able to potentially inhibit the bacterial lipopolysaccharides-induced inflammatory response in RAW 264.7 cells. Results of the cytotoxicity studies revealed that the methanolic extract effectively induced the cytotoxicity at considerably lower concentration in MCF-7, A-549, and COLO-205 cell lines, while the viability of HeLa, CasKi, and HT-29 cells were inhibited at comparatively higher concentrations. Results thus indicated that E. hyssopifolium possessed potent anti-inflammatory and cytotoxic properties. This necessitates further exploration of bioactive phytochemical compounds responsible for these properties for therapeutic applications.

Preclinical evaluation of EpCAM-binding designed ankyrin repeat proteins (DARPins) as targeting moieties for bimodal near-infrared fluorescence and photoacoustic imaging of cancer

PurposeFluorescence-guided surgery (FGS) can play a key role in improving radical resection rates by assisting surgeons to gain adequate visualization of malignant tissue intraoperatively. Designed ankyrin repeat proteins (DARPins) possess optimal pharmacokinetic and other properties for in vivo imaging. This study aims to evaluate the preclinical potential of epithelial cell adhesion molecule (EpCAM)-binding DARPins as targeting moieties for near-infrared fluorescence (NIRF) and photoacoustic (PA) imaging of cancer.MethodsEpCAM-binding DARPins Ac2, Ec4.1, and non-binding control DARPin Off7 were conjugated to IRDye 800CW and their binding efficacy was evaluated on EpCAM-positive HT-29 and EpCAM-negative COLO-320 human colon cancer cell lines. Thereafter, NIRF and PA imaging of all three conjugates were performed in HT-29_luc2 tumor-bearing mice. At 24 h post-injection, tumors and organs were resected and tracer biodistributions were analyzed.ResultsAc2-800CW and Ec4.1-800CW specifically bound to HT-29 cells, but not to COLO-320 cells. Next, 6 nmol and 24 h were established as the optimal in vivo dose and imaging time point for both DARPin tracers. At 24 h post-injection, mean tumor-to-background ratios of 2.60 ± 0.3 and 3.1 ± 0.3 were observed for Ac2-800CW and Ec4.1-800CW, respectively, allowing clear tumor delineation using the clinical Artemis NIRF imager. Biodistribution analyses in non-neoplastic tissue solely showed high fluorescence signal in the liver and kidney, which reflects the clearance of the DARPin tracers.ConclusionOur encouraging results show that EpCAM-binding DARPins are a promising class of targeting moieties for pan-carcinoma targeting, providing clear tumor delineation at 24 h post-injection. The work described provides the preclinical foundation for DARPin-based bimodal NIRF/PA imaging of cancer.

New Thiazole Carboxamide Derivatives as COX Inhibitors: Design, Synthesis, Anticancer Screening, In Silico Molecular Docking, and ADME Profile Studies.

The goal of this work was to create and test a new series of thiazole carboxamide derivatives for their cyclooxygenase (COX) suppressor and anticancer effects. The compounds were characterized using 1H, 13C NMR, and HRMS spectrum analysis, and their selectivity toward COX-1 and COX-2 was assessed using an in vitro COX inhibition assay kit. Cytotoxicity was assessed using an MTS assay against a panel of cancer and normal cell lines. The docking studies were aided by the Prime MM-GBSA method for estimating binding affinities. The density functional theory (DFT) analysis was performed to assess compound chemical reactivity, which was calculated by computing the border orbital energy of both HOMO and LUMO orbitals, as well as the HOMO-LUMO energy gap. For ADME-T analysis, the QiKProp module was employed. Furthermore, using human X-ray crystal structures, molecular docking studies were carried out to discover the probable binding patterns of these drugs within both COX-1 and COX-2 isozymes. The results demonstrated that the most effective compound against the COX-1 enzyme was 2b with an IC50 of 0.239 μM. It also showed potent activity against COX-2 with an IC50 value of 0.191 μM and a selectivity ratio of 1.251. The highest selectivity ratio was 2.766 for compound 2a against COX-2 with an IC50 dose of 0.958 μM relating to the celecoxib ratio of 23.8 and its IC50 against COX-2 of 0.002 μM. Compound 2j also showed good selectivity toward COX-2 (1.507) with an IC50 value of 0.957 μM. All compounds showed negligible cytotoxic activity against the evaluated normal cell lines, and the IC50 values were more than 300 μM, except for compound 2b, whose IC50 values were 203.71 ± 1.89 and 116.96 ± 2.05 μM against LX-2 and Hek293t cell lines, respectively. Moreover, compound 2b showed moderate anticancer activity against COLO205 and B16F1 cancer cell lines with IC50 values of 30.79 and 74.15 μM, respectively.

Antineoplastic Effects of Mucuna pruriens Against Human Colorectal Adenocarcinoma.

Mucuna pruriens (MP) which is commonly known as "Velvet Bean" is an underutilized legume traditionally used to treat Parkinson's disease and male fertility issues. Extracts of MP have also been identified for their antidiabetic, antioxidant, and antineoplastic effects. Commonly, the antioxidant and anticancer properties of a drug are linked together as antioxidants scavenge free radicals and prevent the cellular DNA damage which could result in cancer development. In this investigation, comparative assessment of the anticancer and antioxidant potentials of methanolic seed extracts from two common varieties of MP, Mucuna pruriens var. pruriens (MPP) and Mucuna pruriens var. utilis (MPU) against human colorectal cancer adenocarcinoma cells COLO-205, was carried out. The highest antioxidant potential was recorded with MPP with an IC50 of 45.71μg/ml. The in vitro antiproliferative effects of MPP and MPU on COLO-205 showed an IC50 of 131.1μg/ml and 246.9μg/ml respectively. Our results revealed intervention of the MPP and MPU extracts in growth kinetics of the COLO-205 cells in concomitance with apoptosis induction up to 8.73- and 5.58-folds respectively. The AO/EtBr dual staining and the flow cytometry results also confirmed the better apoptotic efficacy of MPP over MPU. MPP at a concentration of 160μg/ml exhibited highest apoptosis and cell cycle arrest. Furthermore, effect of the seed extracts on p53 expression was investigated by quantitative RT-PCR and a maximum upregulation of 1.12-fold was recorded with MPP.

MRNA-Lipid Nanoparticle (LNP) Delivery of Humanized EpCAM-CD3 Bispecific Antibody Significantly Blocks Colorectal Cancer Tumor Growth.

The epithelial cell adhesion molecule (EpCAM) is often overexpressed in many types of tumors, including colorectal cancer. We sequenced and humanized an EpCAM mouse antibody and used it to develop bispecific EpCAM-CD3 antibodies. Three different designs were used to generate bispecific antibodies such as EpCAM-CD3 CrossMab knob-in-hole, EpCAM ScFv-CD3 ScFv (BITE), and EpCAM ScFv-CD3 ScFv-human Fc designs. These antibody designs showed strong and specific binding to the EpCAM-positive Lovo cell line and T cells, specifically killed EpCAM-positive Lovo cells and not EpCAM-negative Colo741 cells in the presence of T cells, and increased T cells' IFN-gamma secretion in a dose-dependent manner. In addition, transfection of HEK-293 cells with EpCAM ScFv-CD3 ScFv human Fc mRNA-LNPs resulted in antibody secretion that killed Lovo cells and did not kill EpCAM-negative Colo741 cells. The antibody increased IFN-gamma secretion against Lovo target cells and did not increase it against Colo741 target cells. EpCAM-CD3 hFc mRNA-LNP transfection of several cancer cell lines (A1847, C30, OVCAR-5) also demonstrated functional bispecific antibody secretion. In addition, intratumoral delivery of the EpCAM-CD3 human Fc mRNA-LNPs into OVCAR-5 tumor xenografts combined with intravenous injection of T cells significantly blocked xenograft tumor growth. Thus, EpCAM-CD3 hFc mRNA-LNP delivery to tumor cells shows strong potential for future clinical studies.

Synthesis of Selenium Nanoparticles Modified by Quaternary Chitosan Covalently Bonded with Gallic Acid.

Quaternary chitosan derivative with covalently bonded antioxidant (QCG) was used as media for synthesis of selenium nanoparticles (SeNPs). SeNPs were characterized using AFM, TEM, and DLS methods. The data confirmed the formation of stable nanoparticles with a positive charge (34.86-46.73 mV) and a size in the range 119.5-238.6 nm. The antibacterial and fungicidal activity of SeNPs occurred within the range of values for chitosan derivatives. In all cases, the highest activity was against C. albicans (MIC 125 µg/mL). The toxicity of the modified selenium nanoparticles to eukaryotic cells was significantly higher. Among nanoparticle samples, SeNPs that were synthesized at 55 °C demonstrated the highest toxicity against Colo357 and HaCaT cell lines. Based on these results, SeNPs loaded with doxorubicin were obtained. DOX loading efficiency was about 18%. QCG-SeNPs loaded with DOX at a concentration of 1.25 μg/mL inhibited more than 50% of hepatocarcinoma (Colo 357) cells and about 70% of keratinocytes (HaCaT).

EVALUATION OF CYTOTOXIC AND ANTICANCER ACTIVITY OF PANEEYA KSHARA ON COLON CANCER CELL LINES

Kshara is a unique medicinal preparation mentioned in Ayurveda. It is an alkali prepared by burning different medicinal plants. Kshara has cytotoxic activity. It can be used internally and externally. Kshara is used internally as medicine and is called Paneeya kshara. Paneeya kshara is mentioned in the context of Gulma roga. Gulma is a disease with the cardinal feature of a mass per abdomen. Malignant conditions of the colon can be correlated to Gulma. So, this study was planned to prepare the Paneeya kshara mentioned in Gulma disease and study its cytotoxic activity and anticancer activity on colon cancer cell lines. The HEK 293 normal cell line was used to study the cytotoxic activity, and colon cancer cell lines COLO 205, COLO320 and HCT-15 cell lines were used to study the anticancer activity. The cytotoxic activity on HEK293 normal cell lines was good, with an IC50 value of 0.726 µg. IC 50 value for COLO320 cell lines was 0.7263 µg. The effect on HCT -15 and COLO205 cell lines was insignificant, with an IC50 value of more than one. Paneeya kshara showed good cytotoxic action but limited anticancer activity on colon cancer cell lines. This study revealed the potentiality of Paneeya kshara in the management of colon cancer.

Evidence that RXFP4 is located in enterochromaffin cells and can regulate production and release of serotonin.

RXFP4 is a G protein-coupled receptor (GPCR) in the relaxin family. It has recently been recognised that this receptor and its cognate ligand INSL5 may have a role in the regulation of food intake, gut motility, and other functions relevant to metabolic health and disease. Recent data from reporter-mice showed co-location of Rxfp4 and serotonin (5-HT) in the lower gut. We used human single-cell RNA sequence data (scRNASeq) to show that RXFP4 is in a subset of gut enterochromaffin cells that produce 5-HT in humans. We also used RNAScope to show co-location of Rxfp4 mRNA and 5-HT in mouse colon, confirming prior findings. To understand whether RXFP4 might regulate serotonin production, we developed a cell model using Colo320, a human gut-derived immortalised cell line that produces and releases serotonin. Overexpression of RXFP4 in these cells resulted in a constitutive decrease in cAMP levels in both the basal state and in cells treated with forskolin. Treatment of cells with two RXFP4 agonists, INSL5 derived peptide INSL5-A13 and small molecule compound-4, further reduced cAMP levels. This was paralleled by a reduction in expression of mRNA for TPH1, the enzyme controlling the rate limiting step in the production of serotonin. Overexpression of RXFP4 also attenuated the cAMP-induced release of serotonin from Colo320 cells. Together this demonstrates that serotonin producing enterochromaffin cells are the major site of RXFP4 expression in the gut and that RXFP4 can have inhibitory functional impacts on cAMP production as well as TPH1 expression and serotonin release.

Abstract 1377: DCC-3116, a first-in-class selective inhibitor of ULK1/2 kinases and autophagy, synergizes with encorafenib and cetuximab in BRAF V600E mutant colorectal cancer models

Abstract Background: Cancer cells activate autophagy through ULK1/2 kinases as an adaptive stress response (ASR) mechanism to therapies targeting the RTK/RAS/MAPK/PI3K pathways, limiting antitumor response. BRAF signals through the MAPK pathway while EGFR signals upstream through both the MAPK and PI3K pathways, suppressing ULK1/2 kinases and autophagy. Inhibition of mutant BRAF and EGFR reverses this suppression, activating the autophagy ASR1-4. The BRAF V600E mutation occurs in ~10% of colorectal cancer (CRC) patients. Approved treatments for these patients include the BRAF V600E inhibitor encorafenib in combination with the EGFR antibody, cetuximab. Treatment with encorafenib + cetuximab (E+C) is initially successful, but drug resistance develops either through RTK/MAPK resistant mutations or ASR pathways including autophagy. DCC-3116 is a selective, investigational, potent, first-in-class inhibitor of the ULK1/2 kinases in clinical development in combination with targeted therapies that activate the autophagic ASR pathway. Herein we demonstrate ULK1/2 kinases and autophagy are activated upon treatment with E+C. A combination of E+C with DCC-3116 leads to inhibition of ULK1/2-mediated ATG13 phosphorylation (pATG13) and autophagy in vitro. In a PK/PD model, DCC-3116 inhibits E+C induced pATG13 and leads to synergistic tumor growth inhibition (TGI) in a preclinical model of BRAF V600E mutated CRC. Methods: ULK1/2 activity was measured in cells using an ELISA for the ULK substrate phospho-ATG13 (pATG13). Autophagic flux was measured by monitoring mCherry/GFP tagged LC3 in HT-29 and Colo-205 cells. Xenograft models were performed at a CRO. Results: E+C treatment led to the activation of ULK1/2 2-3-fold in BRAF V600E mutated CRC cell lines. DCC-3116 inhibited both E+C -induced and basal pATG13 with IC50 values of 80 nM and 234 nM in HT-29 and Colo-205 cells, respectively. E+C induced autophagic flux ~3-fold which was potently inhibited by DCC-3116 with IC50 values of 65 nM in HT-29 and 40 nM in Colo-205 cells. In a PK/PD model, DCC-3116 inhibited ULK1/2-mediated pATG13 induced by E+C. The combination of DCC-3116 with E+C resulted in greater TGI compared to single agent or the combination of E+C in BRAF V600E models. Conclusions: These preclinical data demonstrate that BRAF inhibitors in combination with EGFR blockade such as E+C activate ULK-mediated autophagy as an ASR resistance mechanism which can be inhibited by DCC-3116, providing the rationale to study the combination of DCC-3116 with E+C in BRAF V600E mutated CRC patients. DCC-3116 is currently in a Phase 1 clinical trial in patients with advanced solid tumors. (NCT04892017).

Abstract 1626: Tumors driven by oncogene amplified extrachromosomal DNA (ecDNA) demonstrate enhanced sensitivity to cell cycle checkpoint kinase 1 (CHK1) inhibition

Abstract Patients whose tumors harbor oncogene amplification on extrachromosomal DNA (ecDNA) fail to respond to targeted or immune therapy and have poor prognosis. ecDNA are cancer-specific circular fragments of genomic DNA engendered with unique properties, including open chromatin architecture associated with hyper transcription and a predilection for structural variation. In addition, ecDNA+ tumors have tremendous copy number heterogeneity mediated through acentric, non-Mendelian segregation. These properties afford ecDNA+ tumors with unparalleled genomic plasticity permitting circumvention of therapeutic pressure. However, these features also confer heightened levels of DNA replication stress (RS), and we have found that ecDNA amplified tumor cells are hyper-reliant on CHK1, a master regulator of the cellular RS response. We identified CHK1 as an ecDNA essential target in a CRISPR genetic screen using a methotrexate-induced ecDNA amplification model in HeLa cancer cells. ecDNA-dependent cell fitness assessment of CHK1 was confirmed using a flow cytometry-based CRISPR competition assay. The target was further validated using a CHK1 inhibitor (CHK1i) tool compound in MYC-amplified COLO320 isogenic cell lines that demonstrated a 10-fold enhanced cytotoxicity in the ecDNA amplified setting.Based on these results, we developed a highly potent, selective, and orally bioavailable CHK1i optimized as an ecDNA-directed therapeutic (ecDTx). An advanced lead, BBI-cmpd1, robustly induced RS biomarkers (e.g., pRPA) in ecDNA+ COLO320 tumor cells as compared to matched chromosomally-amplified (ecDNA-) COLO320 cells, consistent with the increased reliance on CHK1 to manage elevated RS in the ecDNA amplified setting. BBI-cmpd1 demonstrated potent anti-proliferative activity against a panel of ecDNA+ oncogene amplified tumor lines as compared to non-amplified lines demonstrating oncogene and indication agnostic efficacy in ecDNA-based tumors. Oral administration of BBI-cmpd1 resulted in on-target activity against CHK1 and anti-tumor activity in an ecDNA oncogene amplified tumor model in vivo. These findings support the clinical utility of potent, selective, and oral CHK1i to address the significant unmet need driven by ecDNA oncogene amplified cancers. Citation Format: Sudhir Chowdhry, Snezana Milutinovic, Edison Tse, Salvador Garcia, Dean Perusse, Melissa Ritland, Juyeon Ko, Deepti Wilkinson, Kristen Turner, Auzon Steffy, Joshua Plum, Ben Norman, AnnMarie Pferdekamper, Todd Meyer, Debbie Liao, Rachelle Elsdon, Joshua Lange, Anthony Pinkerton, Ryan Hansen, Christian Hassig, Shailaja Kasibhatla. Tumors driven by oncogene amplified extrachromosomal DNA (ecDNA) demonstrate enhanced sensitivity to cell cycle checkpoint kinase 1 (CHK1) inhibition [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 1626.

Abstract 418: Elacridar potentiates sunitinib efficacy in colorectal cancer models

Abstract For unresectable metastatic colorectal cancer (mCRC), the main treatment strategy is combination chemotherapy, targeted therapy, or immunotherapy. Long-term treatment benefit of these therapies is limited to a small subgroup of patients. To improve treatment outcome, many tyrosine kinase inhibitors (TKIs) have been clinically tested in patients with mCRC. Most failed already in early-phase clinical trials due to intrinsic resistance. ATP Binding Cassette Transporter (ABC)B1- and ABCG2-transporters are known contributors to treatment resistance in mCRC. Therefore, we evaluated whether inhibition of these transporters could potentiate the efficacy of TKIs in colorectal cancer (CRC) models. CRC cell lines, HT29, COLO320 and CACO2 and newly established patient-derived tumor organoids (PDTOs) from mCRC biopsies were first analyzed for the presence of ABCB1 and ABCG2 expression. Models that expressed ABCB1 or ABCG2 were pre-incubated with a non-toxic dose of elacridar, a potent transporter inhibitor, and exposed to a concertation range of three TKIs; sunitinib, cediranib and osimertinib. Next, we measured intracellular drug concentrations using LC/MS-MS one hour after TKI exposure (20 µM) with or without pre-incubation of elacridar. Using the fluorescent properties of sunitinib, we performed live-cell imaging to compare intracellular sunitinib distribution in cells that were or were not pre-incubated with elacridar. ABCB1 and ABCG2 expression was observed in all CRC models except for HT29 cells, which did not express ABCB1. Pre-incubation with elacridar significantly improved sunitinib efficacy in CRC cells. The IC50 decreased from 2.15 µM to 1.17 µM (p = 0.04), from 1.32 µM to 0.44 µM (p = 0.03), and from 4.94 µM to 3.43 µM (p = 0.03) for HT29, COLO320 and CACO2 cells respectively. This effect was not observed for cediranib and osimertinib. Although not significant, for both PDTO models tested, the IC50 for sunitinib decreased from 8.04 µM to 3.29 µM and from 6.93 µM to 4.16 µM. Intracellular sunitinib concentrations in the tested CRC cell models after sunitinib exposure ranged from 2.46 mM to 4.13 mM without elacridar and from 0.38 mM µM to 1.31 mM when pre-incubated with elacridar (p < 0.01). Live-cell imaging of CRC cells revealed a different pattern of intracellular sunitinib accumulation with and without pre-incubation with elacridar. Inhibition of multi-drug resistance proteins ABCB1 and ABCG2 with the clinically tested selective transporter inhibitor elacridar potentiates sunitinib efficacy in CRC models. Interestingly, this was accompanied with reduced intracellular sunitinib accumulation. Further research in mCRC PDTOs is warranted to confirm whether this mechanism is involved in intrinsic TKI resistance and therefore contribute to the early-phase failure of clinical trials testing TKIs for mCRC. Citation Format: Dennis Poel, Kirti K. Iyer, Bob van Gasteren, Beau Dagniaux, Erik van den Hombergh, Nielka P. van Erp, Daniele V. Tauriello, Henk M. Verheul. Elacridar potentiates sunitinib efficacy in colorectal cancer models [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 418.

Assessment of metabolic stability, pharmacokinetics by LC-MS/MS and establishment of safe dose of IMID-2, a novel anticancer molecule under drug discovery.

Pyruvate kinase (PK) M2 activators ramp up glycolysis in cancer cells leading to a reversal of the Warburg effect in cancer cells. A promising PKM2 activator molecule IMID-2 developed by National Institute of Pharmaceutical Education & Research, Ahmedabad (NIPER-A) showed promising anticancer activity against MCF-7 and COLO-205 cell lines which represent breast and colon cancer. Its physicochemical properties like solubility, ionization constant, partition coefficient, and distribution constant have already been established. Its metabolic pathway is also well established through in vitro and in vivo metabolite profiling and reported previously. In this study, we have evaluated the metabolic stability of IMID-2 using (LC-MS/MS and investigated the safety aspect of the molecule through an acute oral toxicity study. In vivo studies in rats confirmed that the molecule is safe even at the dose level of 175 mg/kg. Furthermore, the pharmacokinetic study of IMID-2 was also carried out by LC-MS/MS to understand its ADME profile. The molecule was found to have promising bioavailability through the oral route. This research work is thus another step toward drug-likeness testing of this promising anti-cancer molecule. The molecule can be considered to be a potential anti-cancer lead based on the earlier report substantiated by current findings.

Design, Synthesis and In Vitro Biological Activity of Novel C-7 Methylene Congeners of Furanoallocolchicinoids.

A series of novel heterocyclic colchicine derivatives bearing a C-7 methylene fragment were synthesized via Wittig, Horner-Wadsworth-Emmons and Nenajdenko-Shastin olefination approaches. The in vitro biological activities of the most promising compounds were investigated using MTT assays and cell cycle analyses. Compounds with an electron withdrawing group on the methylene fragment exhibited substantial antiproliferative activity towards COLO-357, BxPC-3, HaCaT, PANC-1 and A549 cell lines. The spatial orientation of the substituent at the double bond significantly influenced its biological activity.

Design, Synthesis, and Anticancer Evaluation of Novel Tetracaine Hydrazide-Hydrazones.

Tetracaine is an ester derivative used as a local anesthetic molecule. In this study, a series of novel Tetracaine derivatives bearing hydrazide-hydrazone moiety were designed, synthesized, and evaluated for anticancer activity. The structures of these compounds were characterized by spectral (1H NMR,13C NMR, FT-IR, and HRMS analyses) methods. All synthesized compounds were screened for anticancer activity against two different human cancer cell lines (Colo-205 and HepG2). Among the synthesized molecules, compounds 2f and 2m showed the most potent anticancer activity against the Colo-205 cell line (IC50 = 50.0 and 20.5 μM, respectively). Compounds 2k, 2p, and 2s demonstrated the best anticancer activity against the HepG2 cell line (IC50 = 30.5, 35.9, and 20.8 μM, respectively). mRNA transcription levels of Bax and caspase-3 genes were determined by real-time polymerase chain reaction (qRT-PCR) analysis of both Colo-205 and HepG2 cell lines. Doxorubicin was used as a positive sensitivity reference standard. qRT-PCR analysis showed that there was a time-dependent rise in the expression levels of Bax and Caspase 3 on apoptosis. Inhibition of apoptotic proteins PI3K, Akt, PTEN, pPTEN, FoXO1, FoXO3a, TXNIP, and p27 was investigated in Colo-205 and HepG2 cells treated with compounds 2f, 2m, 2k, 2p, and 2s by using Western blotting.

Synthesis and biological evaluation of benzamide-chalcone hybrids as potential c-Metkinase and COX-2 inhibitors.

c-Met kinase and cyclooxygenase 2 (COX-2) enzymes are two significant targets in tumor progression. Chalcone and benzamide moieties were combined using molecular hybridization to assess their potential as c-Met kinase and COX-2 inhibitors. 4-Methylbenzamide and 4-chlorobenzamide chalcone analogs were synthesized, characterized, and evaluated for antiproliferative activity on Michigan Cancer Foundation-7 (MCF-7), HT-29, MDA-MB-231, COLO-205, and A549 cell lines by sulforhodamine-B stain (SRB) assay. Following the SRB assay, compounds were evaluated for their c-Met kinase and COX-2 inhibitory potential. All compounds inhibited COX-2 with half-maximal inhibitory concentration (IC50 ) <10 µM. Compounds 7h, 7i, 7j, 8f, and 8j inhibited c-Met with IC50 <10 µM. Compound 7h was evaluated for its long-term antiproliferative and anti-migratory effects by colony formation and wound healing assay. It exerted these effects in a concentration-dependent manner. Compounds 7j and 8j were further evaluated for in vitro antiangiogenic effects. Compound 7j exhibited moderate antiangiogenic effect while compound 8j exhibited strong effect. Compounds 7h, 7i, 7j, 8f, and 8j were evaluated for the serum protein binding, using the in vitro bovine serum albumin binding assay. The results indicated that the tested compounds bind to bovine serum albumin (BSA) and can be further explored by other studies.

Phytochemical Analysis and Anticancer Properties of Drimia maritima Bulb Extracts on Colorectal Cancer Cells.

Cancer is a worldwide health problem and is the second leading cause of death after heart disease. Due to the high cost and severe side effects associated with chemotherapy treatments, natural products with anticancer therapeutic potential may play a promising role in anticancer therapy. The purpose of this study was to investigate the cytotoxic and apoptotic characteristics of the aqueous Drimia maritima bulb extract on Caco-2 and COLO-205 colorectal cancer cells. In order to reach such a purpose, the chemical composition was examined using the GC-MS method, and the selective antiproliferative effect was determined in colon cancer cell lines in normal gingival fibroblasts. The intracellular ROS, mitochondrial membrane potential, and gene expression changes in selected genes (CASP8, TNF-α, and IL-6 genes) were assessed to determine the molecular mechanism of the antitumor effect of the extract. GC-MS results revealed the presence of fifty-seven compounds, and Proscillaridin A was the predominant secondary metabolite in the extract. The IC50 of D. maritima bulb extract on Caco-2, COLO-205, and the normal human gingival fibroblasts were obtained at 0.9 µg/mL, 2.3 µg/mL, and 13.1 µg/mL, respectively. The apoptotic effect assay indicated that the bulb extract induced apoptosis in both colon cancer cell lines. D. maritima bulb extract was only able to induce statistically significant ROS levels in COLO-205 cells in a dose-dependent manner. The mitochondrial membrane potential (MMP) revealed a significant decrease in the MMP of Caco-2 and COLO-205 to various concentrations of the bulb extract. At the molecular level, RT-qPCR was used to assess gene expression of CASP8, TNF-α, and IL-6 genes in Caco-2 and COLO-205 cancer cells. The results showed that the expression of pro-inflammatory genes TNF-α and IL-6 were upregulated. The apoptotic initiator gene CASP8 was also upregulated in the Caco-2 cell line and did not reach significance in COLO-205 cells. These results lead to the conclusion that D. maritima extract induced cell death in both cell lines and may have the potential to be used in CRC therapy in the future.

The Assessment of the Phototoxic Action of Chlortetracycline and Doxycycline as a Potential Treatment of Melanotic Melanoma-Biochemical and Molecular Studies on COLO 829 and G-361 Cell Lines.

Melanoma is still one of the most dangerous cancers. New methods of treatment are sought due to its high aggressiveness and the relatively low effectiveness of therapies. Tetracyclines are drugs exhibiting anticancer activity. Previous studies have also shown their activity against melanoma cells. The possibility of tetracycline accumulation in pigmented tissues and the increase in their toxicity under the influence of UVA radiation creates the possibility of developing a new anti-melanoma therapy. This study aimed to analyze the phototoxic effect of doxycycline and chlortetracycline on melanotic melanoma cells COLO 829 and G-361. The results indicated that tetracycline-induced phototoxicity significantly decreased the number of live cells by cell cycle arrest as well as a decrease in cell viability. The simultaneous exposure of cells to drugs and UVA caused the depolarization of mitochondria as well as inducing oxidative stress and apoptosis. It was found that the combined treatment activated initiator and effector caspases, caused DNA fragmentation and elevated p53 level. Finally, it was concluded that doxycycline demonstrated a stronger cytotoxic and phototoxic effect. UVA irradiation of melanoma cells treated with doxycycline and chlortetracycline allows for the reduction of therapeutic drug concentrations and increases the effectiveness of tested tetracyclines.

Properties of Linum usitatissimum Seed Aqueous Extract Against Colon Cancer Cell Lines

Background: Colon cancer has been a rising health concern in the modern world for quite some time. Considering the harmful side effects of chemotherapy, the most common treatment for this cancer, new treatment methods are required to lessen the side effects of treatment on patients. Traditional and herbal medicine can help in this effort. Objectives: Our study investigated the antiproliferative and pro-apoptotic effects of Linum usitatissimum seed aqueous extract on colon cancer and the expression of apoptotic genes compared to normal cell lines to reduce chemotherapy's harmful and irreversible side effects on colon cancer patients. Methods: MTT assay was used to determine the cytotoxicity of our extract on LS174T and COLO205 cells. Cancer and normal cell lines were treated with 2, 4, 6, and 8 mg/mL of LU extract for 24 and 48 hours. RNA extraction was performed, and mRNAs were reverse transcribed to cDNAs using RT-PCR. qPCR was then performed to assess the expression of BAX and BAD genes. Results: MTT assay concluded that this extract has a good cytotoxic effect on LS174t and COLO205 cell lines and can decrease cell viability, while no significant decrease in cell viability was observed in normal cells. For qPCR assay, the cells were treated with 0.5, 1, and 2 mg/mL of LU extract for two days. The qPCR analysis revealed that the expression of pro-apoptotic BAX and BAD was significantly decreased following treatment with our extract, while the expression of these genes showed no significant changes in normal cell lines after the same treatment. Conclusions: These results show that the components within L. usitatissimum seed aqueous extract have antiproliferative and pro-apoptotic properties. This extract can successfully decrease the population of LS174t and COLO205 cells while increasing the expression of pro-apoptotic BAX and BAD in these cell lines. The results also demonstrate that normal non-cancerous cells are unharmed by this extract. These results hint at some potential anti-cancer properties of L. usitatissimum seed aqueous extract.

Isolation of the Lanostane Triterpenes Pholiols L-S from Pholiota populnea and Evaluation of Their Antiproliferative and Cytotoxic Activities.

Pholiols L-S (1−8), eight undescribed triterpenes were isolated from the sporocarps of the mushroom Pholiota populnea. Various chromatographic techniques, such as open column chromatography, flash chromatography, gel filtration, preparative thin layer chromatography, and HPLC, were applied to purify the compounds. The structure elucidation was carried out by spectroscopic analysis, including 1D (1H NMR and 13C JMOD) and 2D NMR (1H-1H COSY, HSQC, HMBC and NOESY) and HRESIMS experiments. The isolated compounds had lanostane (1−7) or trinorlanostane (8) skeletons; all of them were substituted with 3-hydroxy-3-methylglutaroyl group or its 6-methyl ester. Five compounds (1, 2, 4, 6, and 8) were investigated for their antiproliferative and cytotoxic activity in vitro by MTT assay on breast cancer (MCF-7), human colon adenocarcinoma (sensitive Colo 205, and resistant Colo 320), non-small cell lung cancer (A549), and human embryonic lung fibroblast (MRC-5) cell lines. Pholiols M (2) and O (4) showed antiproliferative activity against the MCF-7 cell line with IC50 of 2.48 and 9.95 µM, respectively. These compounds displayed tumor cell selectivity on MCF-7 cells with SI values of >40 (2) and 4.3 (4), but they did not show a cytotoxic effect, proving their action exclusively on tumor cell proliferation. Pholiols L (1) and Q (8) were found to have selective cytotoxicity on drug resistant cells in comparison to their effects on Colo320 and Colo205 cells [relative resistance values 0.84 (1) and 0.62 (8)].

In Vitro and In Silico Biological Studies of 4-Phenyl-2-quinolone (4-PQ) Derivatives as Anticancer Agents

Our previous study found that 2-phenyl-4-quinolone (2-PQ) derivatives are antimitotic agents, and we adopted the drug design concept of scaffold hopping to replace the 2-aromatic ring of 2-PQs with a 4-aromatic ring, representing 4-phenyl-2-quinolones (4-PQs). The 4-PQ compounds, whose structural backbones also mimic analogs of podophyllotoxin (PPT), maybe a new class of anticancer drugs with simplified PPT structures. In addition, 4-PQs are a new generation of anticancer lead compounds as apoptosis stimulators. On the other hand, previous studies showed that 4-arylcoumarin derivatives with 5-, 6-, and 7-methoxy substitutions displayed remarkable anticancer activities. Therefore, we further synthesized a series of 5-, 6-, and 7-methoxy-substituted 4-PQ derivatives (19-32) by Knorr quinoline cyclization, and examined their anticancer effectiveness. Among these 4-PQs, compound 22 demonstrated excellent antiproliferative activities against the COLO205 cell line (50% inhibitory concentration (IC50) = 0.32 μM) and H460 cell line (IC50 = 0.89 μM). Furthermore, we utilized molecular docking studies to explain the possible anticancer mechanisms of these 4-PQs by the docking mode in the colchicine-binding pocket of the tubulin receptor. Consequently, we selected the candidate compounds 19, 20, 21, 22, 25, 27, and 28 to predict their absorption, distribution, metabolism, excretion, and toxicity (ADMET) profiles. Pharmacokinetics (PKs) indicated that these 4-PQs displayed good drug-likeness and bioavailability, and had no cardiotoxic side effects or carcinogenicity, but we detected risks of drug-drug interactions and AMES toxicity (mutagenic). However, structural modifications of these 4-PQs could improve their PK properties and reduce their side effects, and their promising anticancer activities attracted our attention for further studies.