Anticancer Activity of Electron-Deficient Metal Complexes against Colorectal Cancer in vitro Models

An evaluation of the in vitro cytotoxicity of nine electron-deficient half-sandwich metal complexes towards two colorectal cancer cell lines (HCT116 p53+/+, HCT116 p53−/−) and one normal prostate cell line (PNT2) is presented herein. Three complexes were found to be equally cytotoxic towards both colorectal cancer cell lines, suggesting a p53-independent mechanism of action. These complexes are 12 to 34× more potent than cisplatin against HCT116 p53+/+ and HCT116 p53−/− cells. Furthermore, they were found to exhibit little or no cytotoxicity towards PNT2 normal cells, with selectivity ratios greater than 50. To gain an insight into the potential mechanisms of action of the most active compounds, their effects on the expression levels of a panel of genes were measured using qRT-PCR against treated HCT116 p53+/+ and HCT116 p53−/− cells, and cell-cycle analysis was carried out.     

Molecular Mechanisms and Tumor Biological Aspects of 5-Fluorouracil Resistance in HCT116 Human Colorectal Cancer Cells

5-Fluorouracil (5-FU) is a cornerstone drug used in the treatment of colorectal cancer (CRC). However, the development of resistance to 5-FU and its analogs remain an unsolved problem in CRC treatment. In this study, we investigated the molecular mechanisms and tumor biological aspects of 5-FU resistance in CRC HCT116 cells. We established an acquired 5-FU-resistant cell line, HCT116R^F10. HCT116R^F10 cells were cross-resistant to the 5-FU analog, fluorodeoxyuridine. In contrast, HCT116R^F10 cells were collaterally sensitive to SN-38 and CDDP compared with the parental HCT16 cells. Whole-exome sequencing revealed that a cluster of genes associated with the 5-FU metabolic pathway were not significantly mutated in HCT116 or HCT116R^F10 cells. Interestingly, HCT116R^F10 cells were regulated by the function of thymidylate synthase (TS), a 5-FU active metabolite 5-fluorodeoxyuridine monophosphate (FdUMP) inhibiting enzyme. Half of the TS was in an active form, whereas the other half was in an inactive form. This finding indicates that 5-FU-resistant cells exhibited increased TS expression, and the TS enzyme is used to trap FdUMP, resulting in resistance to 5-FU and its analogs.     

Potency and Selectivity Optimization of Tryptophanol-Derived Oxazoloisoindolinones: Novel p53 Activators in Human Colorectal Cancer

To search for novel p53 activators, four series of novel (S)- and (R)-tryptophanol-derived oxazoloisoindolinones were synthesized in a straightforward manner and their antiproliferative activity was evaluated in the human colorectal cancer HCT116 cell line. Structural optimization of the hit compound SLMP53-1 led to the identification of a (R)-tryptophanol-derived isoindolinone that was found to be six-fold more active, with increased selectivity for HCT116 cells with p53 and with low toxicity in normal cells. Binding studies with MDM2 showed that the antiproliferative activity of tryptophanol-derived isoindolinones does not involve inhibition of the main negative regulator of the p53 protein. Molecular docking simulations showed that although these molecules establish hydrophobic interactions with MDM2, they do not possess the required features to bind MDM2.     

Binding of the Phage Display Derived Peptide CaIX-P1 on Human Colorectal Carcinoma Cells Correlates with the Expression of Carbonic Anhydrase IX

Phage display represents an attractive screening strategy for the identification of novel, specific binding ligands that could be used for tumor targeting. Recently, a new peptide (CaIX-P1) with affinity for human carbonic anhydrase IX (CAIX) was identified and evaluated. The aim of the present study is to characterize the properties of CaIX-P1 for targeting human colorectal carcinoma and investigate the correlation of peptide binding with the expression of carbonic anhydrase IX. Human colorectal carcinoma HCT116 and HT29 cells were investigated for CAIX expression using Western Blot analysis. Binding and competition studies of ¹²⁵I-radiolabeled CaIX-P1 were performed on HCT116 cells in vitro. FACS analysis and fluorescence microscopy studies were carried out after cell incubation with fluorescein-labeled CaIX-P1 and rhodamine-labeled anti-human CAIX-mAb. Our studies revealed an enhanced in vitro expression of carbonic anhydrase IX in HCT116 and HT29 cells with increasing cell density. Binding of ¹²⁵I-labeled-CaIX-P1 on HCT116 cells increased with increasing cell density and correlated to the CAIX expression. FACS analysis demonstrated a correlation of cell labeling between FITC-CaIX-P1 and rhodamine-labeled anti-CAIX-mAb in both HCT116 and HT29 cells. The results of our study indicate that the phage display identified peptide CaIX-P1 might be an attractive candidate for the development of a ligand targeting CAIX in colorectal cancer.     

Synthesis and Anticancer Properties of Oxazepines Related to Azaisoerianin and IsoCoQuines

In this article, we report the synthesis and biological properties of a series of novel oxazepines related to isoCA-4 having significant antitumor properties. Among them, three oxazepin-9-ol derivatives display a nanomolar or a sub-nanomolar cytotoxicity level against five human cancer cell lines (HCT116, U87, A549, MCF7, and K562). It was demonstrated that the lead compound in this series inhibits tubulin assembly with an IC₅₀ value of 1 μM and totally arrests the cellular cycle in the G2/M phase at the low concentration of 5 nM in HCT116 and K562 cells. Molecular modeling studies perfectly corroborates these promising results.     

c-Myc Protein Level Affected by Unsymmetrical Bisacridines Influences Apoptosis and Senescence Induced in HCT116 Colorectal and H460 Lung Cancer Cells

Unsymmetrical bisacridines (UAs) are highly active antitumor compounds. They contain in their structure the drugs previously synthesized in our Department: C-1311 and C-1748. UAs exhibit different properties than their monomer components. They do not intercalate to dsDNA but stabilize the G-quadruplex structures, particularly those of the MYC and KRAS genes. Since MYC and KRAS are often mutated and constitutively expressed in cancer cells, they can be used as therapeutic targets. Herein, we investigate whether UAs can affect the expression and protein level of c-Myc and K-Ras in HCT116 and H460 cancer cells, and if so, what are the consequences for the UAs-induced cellular response. UAs did not affect K-Ras, but they strongly influenced the expression and translation of the c-Myc protein, and in H460 cells, they caused its full inhibition. UAs treatment resulted in apoptosis, as confirmed by the morphological changes, the presence of sub-G1 population and active caspase-3, cleaved PARP, annexin-V/PI staining and a decrease in mitochondrial potential. Importantly, apoptosis was induced earlier and to a greater extent in H460 compared to HCT116 cells. Moreover, accelerated senescence occurred only in H460 cells. In conclusion, the strong inhibition of c-Myc by UAs in H460 cells may participate in the final cellular response (apoptosis, senescence).     

Design and Synthesis of DNA‐Interactive β‐Carboline–Oxindole Hybrids as Cytotoxic and Apoptosis‐Inducing Agents

A new series of (E)‐3‐[(1‐aryl‐9H‐pyrido[3,4‐b]indol‐3‐yl)methylene]indolin‐2‐one hybrids were synthesized and evaluated for their in vitro cytotoxic activity against a panel of selected human cancer cell lines, namely, HCT‐15, HCT‐116, A549, NCI‐H460, and MCF‐7, including HFL. Among the tested compounds, (E)‐1‐benzyl‐5‐bromo‐3‐{[1‐(2,5‐dimethoxyphenyl)‐9H‐pyrido[3,4‐b]indol‐3‐yl]methylene}indolin‐2‐one ( 10 s ) showed potent cytotoxicity against HCT‐15 cancer cells with an IC₅₀ value of 1.43±0.26 μm and a GI₅₀ value of 0.89±0.06 μm . Notably, induction of apoptosis by 10 s on the HCT‐15 cell line was characterized by using different staining techniques, such as acridine orange/ethidium bromide (AO/EB) and DAPI. Further, to understand the mechanism of anticancer effects, various assays such as annexin V‐FITC/PI, DCFDA, and JC‐1were performed. The flow cytometric analysis revealed that compound 10 s arrests the HCT‐15 cancer cells at the G0/G1 phase of the cell cycle. Additionally, western blot analysis indicated that treatment of 10 s on HCT‐15 cancer cells led to decreased expression of anti‐apoptotic Bcl‐2 and increased protein expression of both pro‐apoptotic Bax and caspase‐3, ‐8, and ‐9, and cleaved PARP with reference to actin. Next, a clonogenic assay revealed the inhibition of colony formation in HCT‐15 cancer cells by 10 s in a dose‐dependent manner. Moreover, upon testing on normal human lung cells (HFL), the compounds were observed to be safer with a low toxicity profile. In addition, viscosity and molecular‐docking studies showed that compound 10 s has typical intercalation with DNA.     

Spontaneous γH2AX Foci in Human Solid Tumor-Derived Cell Lines in Relation to p21WAF1 and WIP1 Expression

Phosphorylation of H2AX on Ser139 (γH2AX) after exposure to ionizing radiation produces nuclear foci that are detectable by immunofluorescence microscopy. These so-called γH2AX foci have been adopted as quantitative markers for DNA double-strand breaks. High numbers of spontaneous γH2AX foci have also been reported for some human solid tumor-derived cell lines, but the molecular mechanism(s) for this response remains elusive. Here we show that cancer cells (e.g., HCT116; MCF7) that constitutively express detectable levels of p21^WAF1 (p21) exhibit low numbers of γH2AX foci (<3/nucleus), whereas p21 knockout cells (HCT116p21−/−) and constitutively low p21-expressing cells (e.g., MDA-MB-231) exhibit high numbers of foci (e.g., >50/nucleus), and that these foci are not associated with apoptosis. The majority (>95%) of cells within HCT116p21−/− and MDA-MB-231 cultures contain high levels of phosphorylated p53, which is localized in the nucleus. We further show an inverse relationship between γH2AX foci and nuclear accumulation of WIP1, an oncogenic phosphatase. Our studies suggest that: (i) p21 deficiency might provide a selective pressure for the emergence of apoptosis-resistant progeny exhibiting genomic instability, manifested as spontaneous γH2AX foci coupled with phosphorylation and nuclear accumulation of p53; and (ii) p21 might contribute to positive regulation of WIP1, resulting in dephosphorylation of γH2AX.     

Defective Autophagosome Formation in p53-Null Colorectal Cancer Reinforces Crocin-Induced Apoptosis

Crocin, a bioactive molecule of saffron, inhibited proliferation of both HCT116 wild-type and HCT116 p53−/− cell lines at a concentration of 10 mM. Flow cytometric analysis of cell cycle distribution revealed that there was an accumulation of HCT116 wild-type cells in G₁ (55.9%, 56.1%) compared to the control (30.4%) after 24 and 48 h of crocin treatment, respectively. However, crocin induced only mild G2 arrest in HCT116 p53−/− after 24 h. Crocin induced inefficient autophagy in HCT116 p53−/− cells, where crocin induced the formation of LC3-II, which was combined with a decrease in the protein levels of Beclin 1 and Atg7 and no clear p62 degradation. Autophagosome formation was not detected in HCT116 p53−/− after crocin treatment predicting a nonfunctional autophagosome formation. There was a significant increase of p62 after treating the cells with Bafilomycin A1 (Baf) and crocin compared to crocin exposure alone. Annexin V staining showed that Baf-pretreatment enhanced the induction of apoptosis in HCT116 wild-type cells. Baf-exposed HCT116 p53−/− cells did not, however, show any enhancement of apoptosis induction despite an increase in the DNA damage-sensor accumulation, γH2AX indicating that crocin induced an autophagy-independent classical programmed cell death.     

Anticancer Potency Studies of Coordination Driven Self‐Assembled Arene–Ru‐Based Metalla‐Bowls

New tetranuclear cationic metalla‐bowls 5 – 7 with the general formula [Ru₄(p‐cymene)₄(N∩N)₂(OO∩OO)₂]⁴⁺ (N∩N=2,6‐bis(N‐(4‐pyridyl carbamoyl)pyridine, OO∩OO=2,5‐dihydroxy‐1,4‐benzoquinonato ( 5 ), OO∩OO=5,8‐dioxydo‐1,4‐naphthaquinonato ( 6 ), OO∩OO=hoxonato ( 7 )) were prepared by the reaction of the respective dinuclear ruthenium complexes 2 – 4 with a bispyridine amide donor ligand 1 in methanol in the presence of AgO₃SCF₃.These new molecular metalla‐bowls were fully characterized by analytical techniques including elemental analysis as well as ¹H and ¹³C NMR and HR‐ESI‐MS spectroscopy. The structure of metalla‐bowl 6 was determined from X‐ray crystal diffraction data. A UV/visible study was also carried out for the entire suite of new complexes. As with recent studies of similar arene–Ru complexes, the inhibition of cell growth by metalla‐bowls was established against SK‐hep‐1 (liver cancer), AGS (gastric cancer), and HCT‐15 (colorectal cancer) human cancer cell lines. Inhibition of cell growth by 6 was found to be considerably stronger against all cancer cell lines than the anticancer drugs, doxorubicin and cisplatin. In particular, in colorectal cancer cells, expression of the cancer suppressor genes APC and p53 was increased following exposure to 6 .     

p53 Enhances Artemisia annua L. Polyphenols-Induced Cell Death Through Upregulation of p53-Dependent Targets and Cleavage of PARP1 and Lamin A/C in HCT116 Colorectal Cancer Cells

Plant-derived natural polyphenols exhibit anticancer activity without showing any noticeable toxicities to normal cells. The aim of this study was to investigate the role of p53 on the anticancer effect of polyphenols isolated from Korean Artemisia annua L. (pKAL) in HCT116 human colorectal cancer cells. We confirmed that pKAL induced reactive oxygen species (ROS) production, propidium iodide (PI) uptake, nuclear structure change, and acidic vesicles in a p53-independent manner in p53-null HCT116 cells through fluorescence microscopy analysis of DCF/PI-, DAPI-, and AO-stained cells. The pKAL-induced anticancer effects were found to be significantly higher in p53-wild HCT116 cells than in p53-null by hematoxylin staining, CCK-8 assay, Western blot, and flow cytometric analysis of annexin V/PI-stained cells. In addition, expression of ectopic p53 in p53-null cells was upregulated by pKAL in both the nucleus and cytoplasm, increasing pKAL-induced cell death. Moreover, Western bot analysis revealed that pKAL-induced cell death was associated with upregulation of p53-dependent targets such as p21, Bax and DR5 and cleavage of PARP1 and lamin A/C in p53-wild HCT116 cells, but not in p53-null. Taken together, these results indicate that p53 plays an important role in enhancing the anticancer effects of pKAL by upregulating p53 downstream targets and inducing intracellular cell death processes.     

tert‐Butylcarbamate‐Containing Histone Deacetylase Inhibitors: Apoptosis Induction,Cytodifferentiation, and Antiproliferative Activities in Cancer Cells

Herein we report novel pyrrole‐ and benzene‐based hydroxamates ( 8 , 10 ) and 2′‐aminoanilides ( 9 , 11 ) bearing the tert‐butylcarbamate group at the CAP moiety as histone deacetylase (HDAC) inhibitors. Compounds 8 b and 10 c selectively inhibited HDAC6 at the nanomolar level, whereas the other hydroxamates effected an increase in acetyl‐α‐tubulin levels in human acute myeloid leukemia U937 cells. In the same cell line, compounds 8 b and 10 c elicited 18.4 and 21.4 % apoptosis, respectively (SAHA: 16.9 %), and the pyrrole anilide 9 c displayed the highest cytodifferentiating effect (90.9 %). In tests against a wide range of various cancer cell lines to determine its antiproliferative effects, compound 10 c exhibited growth inhibition from sub‐micromolar (neuroblastoma LAN‐5 and SH‐SY5Y cells, chronic myeloid leukemia K562 cells) to low‐micromolar (lung H1299 and A549, colon HCT116 and HT29 cancer cells) concentrations. In HT29 cells, 10 c increased histone H3 acetylation, and decreased the colony‐forming potential of the cancer cells by up to 60 %.     

Mitochondria-Targeted Triphenylphosphonium Conjugated C-3 Modified Betulin: Synthesis,Antitumor Properties and Mechanism of Action

A series of mitochondria-targeted triphenylphosphonium conjugated C-3 modified betulin were synthesized and evaluated against tumor cells. As a result, a new derivative 13 i , the conjugate of 3-O-(3′-acetylphenylacetate)-betulin with triphenylphosphonium, was identified as the one with the best anti-tumor effect. Conjugate 13 i significantly inhibited HCT116 cells with IC₅₀ at 0.66 μM. While betulin, C-3 modified betulin, and the triphenylphosphonium moiety showed no inhibition of HCT116 cell proliferation at 20 μM. More importantly, 13 i exhibited a more cytotoxic effect against the tumor cell HCT116 than normal cell NCM460. Mode of action studies demonstrated that 13 i induced the G2/M phase cell cycle arrest and apoptosis in HCT116 cells through the mitochondrial pathway. Structure-activity relationship analysis revealed that integration of triphenylphosphonium moiety into the C-28 of betulin can greatly improve cytotoxicity. Appropriate modification on C-3 of the conjugate would improve the selectivity.     

Development of pH‐sensitive nanogels for cancer treatment using crosslinked poly(aspartic acid‐graft‐imidazole)‐block‐poly(ethylene glycol)

pH‐sensitive nanogels (NGs) based on poly(aspartic acid‐graft‐imidazole)‐poly(ethylene glycol) were developed using linear PEG with different molecular weights (2000 and 4000 Da) as crosslinkers. The pH‐sensitive NGs showed reversible size changes during continuously alternating pH changes. The anticancer treatment potential of pH‐sensitive NGs was studied using a model drug, irinotecan (IRI). IRI‐loaded NGs (ILNs) showed different drug release kinetics in acidic versus neutral pH, in addition to pH‐dependent cytotoxicity. Due to its longer crosslinker, ILN 4 (crosslinked with PEG 4000) showed faster IRI release and a greater magnitude of IRI release than ILN 2 (crosslinked with PEG 2000), resulting in greater cytotoxicity against HCT 116 colorectal cancer cells. These pH‐sensitive NGs could potentially be used in cancer treatment by mediating the accumulation and release of IRI from ILNs in the acidic tumor environment and by reducing systemic toxicity due to reversible swelling–shrinkage. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018 , 135, 46268.     

Development of Isatin-Based Schiff Bases Targeting VEGFR-2 Inhibition: Synthesis,Characterization, Antiproliferative Properties,and QSAR Studies

Three sets of isatin-based Schiff bases were synthesized utilizing the molecular hybridization approach. Some of the synthesized Schiff bases show significant to moderate antiproliferative properties against MCF7 (breast), HCT116 (colon), and PaCa2 (pancreatic) cancer cell lines with potency compared to reference drugs 5-fluorouracil (5-FU) and Sunitinib. Among all, compound 17 f (3-((1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)imino)-1-((1-(2-methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-5-methylindolin-2-one) exhibits promising antiproliferative properties against the MCF7 cancer cell line with 2.1-fold more potency than Sunitinib. However, among all the synthesized compounds, three (5-methylisatin derivatives) were the most effective against HCT116 in comparison to 5-FU. Compound 17 f exhibited the highest anti-angiogenic effect on the vasculature as it significantly reduced BV from 43 mm to 2 mm in comparison to 5.7 mm for Sunitinib and flow cytometry supports the arrest of the cell cycle at G1/S phases. In addition, compound 17 f also showed high VEGFR-2 inhibition properties against breast cancer cell lines. Robust 2D-QSAR studies supported the biological data.     

Discovery and Structure–Activity Relationships of N-Aryl 6-Aminoquinoxalines as Potent PFKFB3 Kinase Inhibitors

Energy and biomass production in cancer cells are largely supported by aerobic glycolysis in what is called the Warburg effect. The process is regulated by key enzymes, among which phosphofructokinase PFK-2 plays a significant role by producing fructose-2,6-biphosphate; the most potent activator of the glycolysis rate-limiting step performed by phosphofructokinase PFK-1. Herein, the synthesis, biological evaluation and structure–activity relationship of novel inhibitors of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), which is the ubiquitous and hypoxia-induced isoform of PFK-2, are reported. X-ray crystallography and docking were instrumental in the design and optimisation of a series of N-aryl 6-aminoquinoxalines. The most potent representative, N-(4-methanesulfonylpyridin-3-yl)-8-(3-methyl-1-benzothiophen-5-yl)quinoxalin-6-amine, displayed an IC₅₀ of 14 nm for the target and an IC₅₀ of 0.49 μm for fructose-2,6-biphosphate production in human colon carcinoma HCT116 cells. This work provides a new entry in the field of PFKFB3 inhibitors with potential for development in oncology.     

Preclinical Anticancer Activity of an Electron-Deficient Organoruthenium(II) Complex

Ruthenium compounds have been shown to be promising alternatives to platinum(II) drugs. However, their clinical success depends on achieving mechanisms of action that overcome Pt-resistance mechanisms. Electron-deficient organoruthenium complexes are an understudied class of compounds that exhibit unusual reactivity in solution and might offer novel anticancer mechanisms of action. Here, we evaluate the in vitro and in vivo anticancer properties of the electron-deficient organoruthenium complex [(p-cymene)Ru(maleonitriledithiolate)]. This compound is found to be highly cytotoxic: 5 to 60 times more potent than cisplatin towards ovarian (A2780 and A2780cisR), colon (HCT116 p53+/+ and HCT116 p53−/−), and non-small cell lung H460 cancer cell lines. It shows no cross-resistance and is equally cytotoxic to both A2780 and A2780cisR cell lines. Furthermore, unlike cisplatin, the remarkable in vitro antiproliferative activity of this compound appears to be p53-independent. In vivo evaluation in the hollow-fibre assay across a panel of cancer cell types and subcutaneous H460 non-small cell lung cancer xenograft model hints at the activity of the complex. Although the impressive in vitro data are not fully corroborated by the in vivo follow-up, this work is the first preclinical study of electron-deficient half-sandwich complexes and highlights their promise as anticancer drug candidates.     

Synthesis and Biological Evaluation of CF3Se-Substituted α-Amino Acid Derivatives

Several CF₃Se-substituted α-amino acid derivatives, such as (R)-2-amino-3-((trifluoromethyl)selanyl)propanoates ( 5 a / 6 a ), (S)-2-amino-4-((trifluoromethyl)selanyl)butanoates ( 5 b / 6 b ), (2R,3R)-2-amino-3-((trifluoromethyl)selanyl)butanoates ( 5 c / 6 c ), (R)-2-((S)-2-amino-3-phenylpropanamido)-3-((trifluoromethyl)selanyl)propanoates ( 11 a / 12 a ), and (R)-2-(2-aminoacetamido)-3-((trifluoromethyl)selanyl)propanoates ( 11 b / 12 b ), were readily synthesized from natural amino acids and [Me₄N][SeCF₃]. The primary in vitro cytotoxicity assays revealed that compounds 6 a , 11 a and 12 a were more effective cell growth inhibitors than the other tested CF₃Se-substituted derivatives towards MCF-7, HCT116, and SK-OV-3 cells, with their IC₅₀ values being less than 10 μM for MCF-7 and HCT116 cells. This study indicated the potentials of CF₃Se moiety as a pharmaceutically relevant group in the design and synthesis of novel biologically active molecules.