Human Colon Adenocarcinoma HCT116 Research Articles

Structural Design of Oleogel‐Hydrogel Bigels for Co‐Delivery of Curcumin and Epigallocatechin Gallate with Synergistic Stability and Bioactivity

AbstractBigels are novel biphasic systems containing hydrogel and oleogel structures, which are designed to deliver multiple bioactives for synergistic activity. The current study develops bigels based on a carrageenan hydrogel and a monoglyceride (GMS) oleogel. Results show that the microstructures of bigels are facilely modified by two surface active ingredients (Tween 20 and polyglycerol polyricinoleate), which then influence the microstructures of the bigels. When curcumin and epigallocatechin gallate (EGCG) are delivered simultaneously, the bioactives work synergistically to slow thermal degradation. Simulated digestion tests indicate that the bigel structures can delay the release of the two bioactives in artificial saliva and simulated gastric fluid (SGF), and allow fast release in simulated intestinal fluid (SIF). DPPH and ABTS tests show the digested Tween 20 bigels containing EGCG and curcumin present synergistic antioxidant activity. Anticancer activity is evaluated in human colon adenocarcinoma HCT116, and the two bioactives work cooperatively to lower cell viability. The bioactives released from Tween 20 bigels have higher cytotoxicity than those from polyglycerol polyricinoleate bigels. The results reveal that facile structural design of bigels has the potential to achieve synergistic stability and bioactivity of co‐delivered bioactives, which is meaningful for the development of functional pharmaceutical, cosmetic, and food products.

Metabolism-Guided Optimization of Tryptophanol-Derived Isoindolinone p53 Activators.

For the first time, the pharmacokinetic (PK) profile of tryptophanol-derived isoindolinones, previously reported as p53 activators, was investigated. From the metabolites' identification, performed by liquid chromatography coupled to high resolution tandem mass spectrometry (LC-HRMS/MS), followed by their preparation and structural elucidation, it was possible to identify that the indole C2 and C3 are the main target of the cytochrome P450 (CYP)-promoted oxidative metabolism in the tryptophanol-derived isoindolinone scaffold. Based on these findings, to search for novel p53 activators a series of 16 enantiopure tryptophanol-derived isoindolinones substituted with a bromine in indole C2 was prepared, in yields of 62-89%, and their antiproliferative activity evaluated in human colon adenocarcinoma HCT116 cell lines with and without p53. Structural optimization led to the identification of two (S)-tryptophanol-derived isoindolinones 3.9-fold and 1.9-fold more active than hit SLMP53-1, respectively. Compounds' metabolic stability evaluation revealed that this substitution led to a metabolic switch, with the impact of Phase I oxidative metabolism being minimized. Through differential scanning fluorimetry (DSF) experiments, the most active compound of the series in cell assays led to an increase in the protein melting temperature (Tm) of 10.39 °C, suggesting an effective binding to wild-type p53 core domain.

Inhibition of Transketolase Improves the Prognosis of Colorectal Cancer.

Colorectal cancer (CRC) remains a heavy health burden worldwide. Transketolase (TKT) is a crucial enzyme in the non-oxidative phase of the Pentose Phosphate Pathway (PPP), and is up-regulated in multiple cancer types. However, the role of TKT in the prognosis of CRC remains unclear. We aimed to explore whether TKT expression is altered in CRC, how TKT is associated with the prognosis of CRC, and whether the regulation of TKT might have an impact on CRC. Differentially expressed genes (DEGs) were identified using bioinformatics analysis. TKT expression was examined in the human colon adenocarcinoma tissue microarray and xenografts. Cell viability, proliferation, migration, and apoptosis assays in vitro were applied to evaluate the protumoral effects of TKT on CRC. TKT was found to be a risk factor for the poor prognosis of CRC by bioinformatics analysis among the DEGs. TKT was significantly up-regulated in colon adenocarcinoma tissues compared with normal colon tissues in patients. Moreover, similar results were found in HCT116 and RKO human colon adenocarcinoma xenografts in nude mice. TKT expression was positively associated with advanced TNM stage, positive lymph nodes, and poor 5 or 10-year overall survival of CRC patients. In vitro, inhibition of TKT reduced cell viability, proliferation, and migration, and induced cell apoptosis. In addition, inhibition of TKT decreased the protein levels of NICD and Hes1. In conclusion, high TKT expression was associated with the poor prognosis of CRC patients. The protumoral effects of downregulating TKT may be realized by suppressing the Notch signaling pathway. TKT may be a new prognostic biomarker and therapeutic target for CRC.

Oleanolic acid from black raisins, Vitis vinifera with antioxidant and antiproliferative potentials on HCT 116 colon cancer cell line

Vitis vinifera (black raisin) is commonly used in traditional medicine for the treatment of various ailments. In the present study, anti-oxidative and anti-cancer efficacy of oleanolic acid from ethyl acetate fraction of black raisins was evaluated and oleanolic acid was isolated without using of any chromatographic techniques and subjected to spectral assessment using UV-Vis spectrophotometer, 1 H NMR, 13C NMR, MS and FT-IR for structural confirmation. Antiproliferative efficacy of oleanolic acid against human colon adenocarcinoma HCT-116 cells was assessed using cell viability assay. The minimum inhibitory concentration (IC50) was determined and found to be 40 µg/mL at 48h incubation. Furthermore, antioxidant property of oleanolic acid was analyzed using DPPH method (IC50 is 61.5µg/mL) by compared to standard antioxidants ascorbic acid, gallic acid, pyrogallol and butylated hydroxytoluene. Hence, the present study aims to establish the use of oleanolic acid as a potential therapeutic agent against human colon cancer.

Marine Bacteria from Rocas Atoll as a Rich Source of Pharmacologically Active Compounds.

Rocas Atoll is a unique environment in the equatorial Atlantic Ocean, hosting a large number of endemic species, however, studies on the chemical diversity emerging from this biota are rather scarce. Therefore, the present work aims to assess the metabolomic diversity and pharmacological potential of the microbiota from Rocas Atoll. A total of 76 bacteria were isolated and cultured in liquid culture media to obtain crude extracts. About one third (34%) of these extracts were recognized as cytotoxic against human colon adenocarcinoma HCT-116 cell line. 16S rRNA gene sequencing analyses revealed that the bacteria producing cytotoxic extracts were mainly from the Actinobacteria phylum, including Streptomyces, Salinispora, Nocardiopsis, and Brevibacterium genera, and in a smaller proportion from Firmicutes phylum (Bacillus). The search in the spectral library in GNPS (Global Natural Products Social Molecular Networking) unveiled a high chemodiversity being produced by these bacteria, including rifamycins, antimycins, desferrioxamines, ferrioxamines, surfactins, surugamides, staurosporines, and saliniketals, along with several unidentified compounds. Using an original approach, molecular networking successfully highlighted groups of compounds responsible for the cytotoxicity of crude extracts. Application of DEREPLICATOR+ (GNPS) allowed the annotation of macrolide novonestimycin derivatives as the cytotoxic compounds existing in the extracts produced by Streptomyces BRB-298 and BRB-302. Overall, these results highlighted the pharmacological potential of bacteria from this singular atoll.

Kinetics of Flavoenzyme-Catalyzed Reduction of Tirapazamine Derivatives: Implications for Their Prooxidant Cytotoxicity.

Derivatives of tirapazamine and other heteroaromatic N-oxides (ArN→O) exhibit promising antibacterial, antiprotozoal, and tumoricidal activities. Their action is typically attributed to bioreductive activation and free radical generation. In this work, we aimed to clarify the mechanism(s) of aerobic mammalian cell cytotoxicity of ArN→O performing the parallel studies of their reactions with NADPH:cytochrome P-450 reductase (P-450R), adrenodoxin reductase/adrenodoxin (ADR/ADX), and NAD(P)H:quinone oxidoreductase (NQO1); we found that in P-450R and ADR/ADX-catalyzed single-electron reduction, the reactivity of ArN→O (n = 9) increased with their single-electron reduction midpoint potential (E17), and correlated with the reactivity of quinones. NQO1 reduced ArN→O at low rates with concomitant superoxide production. The cytotoxicity of ArN→O in murine hepatoma MH22a and human colon adenocarcinoma HCT-116 cells increased with their E17, being systematically higher than that of quinones. The cytotoxicity of both groups of compounds was prooxidant. Inhibitor of NQO1, dicoumarol, and inhibitors of cytochromes P-450 α-naphthoflavone, isoniazid and miconazole statistically significantly (p < 0.02) decreased the toxicity of ArN→O, and potentiated the cytotoxicity of quinones. One may conclude that in spite of similar enzymatic redox cycling rates, the cytotoxicity of ArN→O is higher than that of quinones. This is partly attributed to ArN→O activation by NQO1 and cytochromes P-450. A possible additional factor in the aerobic cytotoxicity of ArN→O is their reductive activation in oxygen-poor cell compartments, leading to the formation of DNA-damaging species similar to those forming under hypoxia.

Synthesis, Biological Evaluation, and In Silico Studies of Novel Aminated Xanthones as Potential p53-Activating Agents.

Xanthone scaffold has been regarded as an attractive chemical tool in the search for bioactive molecules with antitumor activity, and in particular two xanthone derivatives, 12-hydroxy-2,2-dimethyl-3,4-dihydro-2H,6H-pyrano [3,2-b]xanthen-6-one (4) and 3,4-dimethoxy-9-oxo-9H-xanthene-1-carbaldehyde (5), were described as a murine double minute 2 (MDM2)-p53 inhibitor and a TAp73 activator, respectively. The xanthone 5 was used as a starting point for the construction of a library of 3,4-dioxygenated xanthones bearing chemical moieties of described MDM2-p53 inhibitors. Eleven aminated xanthones were successfully synthesized and initially screened for their ability to disrupt the MDM2-p53 interaction using a yeast cell-based assay. With this approach, xanthone 37 was identified as a putative p53-activating agent through inhibition of interaction with MDM2. Xanthone 37 inhibited the growth of human colon adenocarcinoma HCT116 cell lines in a p53-dependent manner. The growth inhibitory effect of xanthone 37 was associated with the induction of G1-phase cell cycle arrest and increased protein expression levels of p53 transcriptional targets. These results demonstrated the potential usefulness of coupling amine-containing structural motifs of known MDM2-p53 disruptors into a 3,4-dioxygenated xanthone scaffold in the design of novel and potent p53 activators with antitumor activity and favorable drug-like properties. Moreover, in silico docking studies were performed in order to predict the binding poses and residues involved in the potential MDM2-p53 interaction.

Design, Synthesis, In Vitro Anti-cancer Activity, ADMET Profile and Molecular Docking of Novel Triazolo[3,4-a]phthalazine Derivatives Targeting VEGFR-2 Enzyme.

Extensive studies were reported in the synthesis of several phthalazine derivatives as promising anticancer agents as potent VEGFR-2 inhibitors. Vatalanib (PTK787) was the first anilinophthalazine published derivative as a potent inhibitor of VEGFR. The discovery of vatalanib as a clinical candidate led to the design and synthesis of different anilinophthalazine derivatives as potent inhibitors for VEGFR-2. The objective of present research work is the synthesis of new agents with the same essential pharmacophoric features of the reported and clinically used VEGFR-2 inhibitors (e.g vatalanib and sorafenib). The main core of our molecular design rationale comprised bioisosteric modification strategies of VEGFR-2 inhibitors at four different positions. A correlation between structure and biological activity of our designed phthalazines was established using molecular docking and VEGFR-2 kinase assay. In view of their expected anticancer activity, novel triazolo[3,4-a]phthalazine derivatives 5-6a-o and 3-substituted-bis([1,2,4]triazolo)[3,4-a:4',3'-c]phthalazines 9a-b were designed, synthesized and evaluated for their anti-proliferative activity against two human tumor cell lines HCT-116 human colon adenocarcinoma and MCF-7 breast cancer. It was found that, compound 6o the most potent derivative against both HCT116 and MCF-7 cancer cell lines. Compounds 6o, 6m, 6d and 9b showed the highest anticancer activities against HCT116 human colon adenocarcinoma with IC50 of 7±0.06, 13±0.11, 15±0.14 and 23±0.22 µM respectively while compounds 6o, 6d, 6a and 6n showed the highest anticancer activities against MCF-7 breast cancer with IC50 of 16.98±0.15, 18.2±0.17, 57.54±0.53 and 66.45±0.67 µM respectively. Sorafenib as a highly potent VEGFR-2 inhibitor was used as a reference drug with IC50 of 5.47±0.3 and 7.26±0.3 µM respectively. Nine compounds were further evaluated for their VEGFR-2 inhibitory activity. Compounds 6o, 6m, 6d and 9b emerged as the most active counterparts against VEGFR-2 with IC50 values of 0.1±0.01, 0.15±0.02, 0.28±0.03 and 0.38±0.04 µM, respectively comparable to that of sorafenib (IC50 = 0.1±0.02) µM. Furthermore, molecular docking studies were carried out for all synthesized compounds to investigate their binding pattern and predict their binding affinities towards VEGFR-2 active site. In silico ADMET studies were calculated for the tested compounds. Most of our designed compounds exhibited good ADMET profile. The obtained results showed that, the most active compounds could be useful as a template for future design, optimization, adaptation and investigation to produce more potent and selective VEGFR-2 inhibitors with higher anticancer analogs.

Improving anticancer activity towards colon cancer cells with a new p53-activating agent.

Impairment of the tumour suppressor p53 pathway is a major event in human cancers, making p53 activation one of the most attractive therapeutic strategies to halt cancer. Here, we have identified a new selective p53 activator and investigated its potential as an anticancer agent. Anti-proliferative activity of the (R)-tryptophanol-derived bicyclic lactam SYNAP was evaluated in a range of human cancer cells with different p53 status. The anticancer activity and mechanism of action of SYNAP was studied in two- and three-dimensional models of human colon adenocarcinoma HCT116 cells with wild-type p53 and corresponding p53-null isogenic derivative cells, alone and in combination with known chemotherapeutic agents. SYNAP showed anti-proliferative effect in human cancer cells dependent on p53 status. In HCT116 cells, SYNAP caused p53-dependent growth inhibition, associated with cell cycle arrest and apoptosis, anti-migratory activity and regulation of the expression of p53 transcriptional targets. Data also indicated that SYNAP targeted p53, inhibiting its interaction with its endogenous inhibitors, murine double minute (MDM)2 and MDMX. Moreover, SYNAP sensitized colon cancer cells to the cytotoxic effect of known chemotherapeutic agents. SYNAP did not induce acquired or cross-resistance and re-sensitized doxorubicin-resistant colon cancer cells to chemotherapy. Additionally, SYNAP was non-genotoxic and had low cytotoxicity against normal cells. SYNAP revealed encouraging anticancer activity, either alone or in combination with known chemotherapeutic agents, in colon cancer cells. Apart from its promising application in cancer therapy, SYNAP may provide a starting point for improved p53 activators.

Design, Synthesis, Molecular Docking, and Anticancer Activity of Phthalazine Derivatives as VEGFR-2 Inhibitors.

Novel series of phthalazine derivatives 6-11 were designed, synthesized, and evaluated for their anticancer activity against two human tumor cell lines, HCT-116 human colon adenocarcinoma and MCF-7 breast cancer cells, targeting the VEGFR-2 enzyme. Compounds 7a,b and 8b,c showed the highest anticancer activities against both HCT116 human colon adenocarcinoma cells with IC50 of 6.04 ± 0.30, 13.22 ± 0.22, 18 ± 0.20, and 35 ± 0.45 μM, respectively, and MCF-7 breast cancer cells with IC50 of 8.8 ± 0.45, 17.9 ± 0.50, 25.2 ± 0.55, and 44.3 ± 0.49 μM, respectively, in comparison to sorafenib as reference drug with IC50 of 5.47 ± 0.3 and 7.26 ± 0.3 μM, respectively. Eleven compounds in this series were further evaluated for their inhibitory activity against VEGFR-2, where compounds 7a, 7b, 8c, and 8b also showed the highest VEGFR-2 inhibition with IC50 of 0.11 ± 0.01, 0.31 ± 0.03, 0.72 ± 0.08, and 0.91 ± 0.08 μM, respectively, in comparison to sorafenib as reference ligand with IC50 of 0.1 ± 0.02. Furthermore, molecular docking studies were performed for all synthesized compounds to predict their binding pattern and affinity towards the VEGFR-2 active site, in order to rationalize their anticancer activity in a qualitative way.

DIMP53-1: a novel small-molecule dual inhibitor of p53-MDM2/X interactions with multifunctional p53-dependent anticancer properties.

The transcription factor p53 plays a crucial role in cancer development and dissemination, and thus, p53‐targeted therapies are among the most encouraging anticancer strategies. In human cancers with wild‐type (wt) p53, its inactivation by interaction with murine double minute (MDM)2 and MDMX is a common event. Simultaneous inhibition of the p53 interaction with both MDMs is crucial to restore the tumor suppressor activity of p53. Here, we describe the synthesis of the new tryptophanol‐derived oxazoloisoindolinone DIMP53‐1 and identify its activity as a dual inhibitor of the p53–MDM2/X interactions using a yeast‐based assay. DIMP53‐1 caused growth inhibition, mediated by p53 stabilization and upregulation of p53 transcriptional targets involved in cell cycle arrest and apoptosis, in wt p53‐expressing tumor cells, including MDM2‐ or MDMX‐overexpressing cells. Importantly, DIMP53‐1 inhibits the p53–MDM2/X interactions by potentially binding to p53, in human colon adenocarcinoma HCT116 cells. DIMP53‐1 also inhibited the migration and invasion of HCT116 cells, and the migration and tube formation of HMVEC‐D endothelial cells. Notably, in human tumor xenograft mice models, DIMP53‐1 showed a p53‐dependent antitumor activity through induction of apoptosis and inhibition of proliferation and angiogenesis. Finally, no genotoxicity or undesirable toxic effects were observed with DIMP53‐1. In conclusion, DIMP53‐1 is a novel p53 activator, which potentially binds to p53 inhibiting its interaction with MDM2 and MDMX. Although target‐directed, DIMP53‐1 has a multifunctional activity, targeting major hallmarks of cancer through its antiproliferative, proapoptotic, antiangiogenic, anti‐invasive, and antimigratory properties. DIMP53‐1 is a promising anticancer drug candidate and an encouraging starting point to develop improved derivatives for clinical application.

Che-1 sustains hypoxic response of colorectal cancer cells by affecting Hif-1\u03b1 stabilization

BackgroundSolid tumours are less oxygenated than normal tissues. Consequently, cancer cells acquire to be adapted to a hypoxic environment. The poor oxygenation of solid tumours is also a major indicator of an adverse cancer prognosis and leads to resistance to conventional anticancer treatments. We previously showed the involvement of Che-1/AATF (Che-1) in cancer cell survival under stress conditions. Herein we hypothesized that Che-1 plays a role in the response of cancer cells to hypoxia.MethodsThe human colon adenocarcinoma HCT116 and HT29 cell lines undepleted or depleted for Che-1 expression by siRNA, were treated under normoxic and hypoxic conditions to perform studies regarding the role of this protein in metabolic adaptation and cell proliferation. Che-1 expression was detected using western blot assays; cell metabolism was assessed by NMR spectroscopy and functional assays. Additional molecular studies were performed by RNA seq, qRT-PCR and ChIP analyses.ResultsHere we report that Che-1 expression is required for the adaptation of cells to hypoxia, playing an important role in metabolic modulation. Indeed, Che-1 depletion impacted on HIF-1α stabilization, thus downregulating the expression of several genes involved in the response to hypoxia and affecting glucose metabolism.ConclusionsWe show that Che-1 a novel player in the regulation of HIF-1α in response to hypoxia. Notably, we found that Che-1 is required for SIAH-2 expression, a member of E3 ubiquitin ligase family that is involved in the degradation of the hydroxylase PHD3, the master regulator of HIF-1α stability.

Endophytic fungi associated with Sudanese medicinal plants show cytotoxic and antibiotic potential.

In this study, we isolated 15 endophytic fungi from five Sudanese medicinal plants. Each fungal endophytic strain was identified by sequencing of internal transcribed spacer (ITS) regions of rDNA. Ethyl acetate extracts were prepared from each endophyte cultivated in vitro and tested for their respective antibacterial activities and antiproliferative activities against human cancer cells. Antibacterial screening was carried out against two bacterial strains: Gram-negative Escherichia coli and Gram-positive methicillin-resistant Staphylococcus aureus, by the broth dilution method. Cell viability was evaluated by the MTT procedure after exposure of MCF7 breast cancer cells and HT29 or HCT116 human colon adenocarcinoma cells to each endophytic extract. Of interest, Byssochlamys spectabilis isolated from Euphorbia prostata showed cytotoxicity (IC50 = 1.51 ± 0.2 μgmL(-1)) against MCF7 cells, but had a low effect against HT29 or HCT116 cells (IC50 > 20 μgmL(-1)). Cladosporium cladosporioides 2, isolated from Vernonia amygdalina leaves, showed antiproliferative activities against MCF7 cells (IC50 = 10.5± 1.5 μgmL(-1)) only. On the other hand, B. spectabilis and Alternaria sp. extract had antibacterial activities against the S. aureus strain. The findings of this work revealed that endophytic fungi associated with medicinal plants from Sudan could be considered as an attractive source of new therapeutic compounds.

Modulation of transforming growth factor‑β signaling transducers in colon adenocarcinoma cells induced by staphylococcal enterotoxin B.

Colorectal cancer (CRC) is a notable cause of cancer‑associated mortality worldwide, making it a pertinent topic for the study of cancer and its treatment. Staphylococcal enterotoxin B (SEB), an enterotoxin produced by Staphylococcus aureus, has been demonstrated to exert anticancer and antimetastatic effects due to its ability to modify cell immunity and cellular signaling pathways. In the current study, SEB was investigated, including whether it exerts its growth inhibitory effects on colon adenocarcinoma cells. This may occur through the manipulation of a key tumor growth factor, termed transforming growth factor‑β (TGF‑β), and its signaling pathway transducer, Smad2/3. The human colon adenocarcinoma HCT116 cell line was treated with different concentrations of SEB, and cell number was measured using MTT assay at different treatment times. Smad2/3 RNA expression level was analyzed in untreated or SEB‑treated cells using quantitative polymerase chain reaction, which indicated significant differences between cell viability and Smad2/3 expression levels. SEB effectively downregulated Smad2/3 expression in the HCT116 cells at concentrations of 1 and 2 µg/ml (P=0.0021 and P=0.0017, respectively). SEB concentrations that were effective at inhibiting Smad2/3 expression were correlated with those able to inhibit the proliferation of the cancer cells. SEB inhibited Smad2/3 expression at the mRNA level in a concentration‑ and time‑dependent manner. The present study thus proposed SEB as an agent able to significantly reduce Smad2/3 expression in colon cancer cells, provoking moderate TGF‑β growth signaling and the reduction of tumor cell proliferation.

Enhanced cytotoxicity of prenylated chalcone against tumour cells via disruption of the p53-MDM2 interaction.

Chalcones are naturally occurring compounds with recognized anticancer activity. It was recently shown that the O-prenyl derivative (2) of 2'-hydroxy-3,4,4',5,6'-pentamethoxychalcone (1) had a remarkably increased cytotoxicity against human tumour cells compared to its precursor. With this study, we aimed to investigate the molecular mechanism underlying the improved tumour cytotoxicity of prenylchalcone 2. The impact of chalcones 1 and 2 on p53-MDM2 interaction was investigated using yeast growth-inhibitory and p53 transactivation assays. Their tumour growth-inhibitory effects were assessed on human colon adenocarcinoma HCT116 cell lines with wild-type p53 and its p53-null derivative, followed by analysis of cell cycle and apoptosis. In tumour cells, the activation of a mitochondrial pathway was checked by analysis of reactive oxygen species generation, Bax mitochondrial translocation and cytochrome c release. Additionally, the up-regulation of p53 transcriptional activity was investigated through Western blot analysis of p53 target expression levels, and the disruption of the p53-MDM2 interaction was confirmed by co-immunoprecipitation. The potent cell tumour growth-inhibitory activity of prenylchalcone 2 was associated with the activation of a p53 pathway involving cell cycle arrest and a mitochondria-dependent apoptosis. Furthermore, a correlation between the distinct cytotoxicity of chalcones 1 and 2 and their ability to disrupt the p53-MDM2 interaction was established. This work shows that prenylation is a determinant factor for the enhancement of chalcones tumour cytotoxicity by improving their ability to disrupt the p53-MDM2 interaction. Prenylchalcone 2 represents a starting basis for the design of new p53-MDM2 interaction inhibitors with improved antitumor properties.

P-Methoxycinnamic acid, an active phenylpropanoid induces mitochondrial mediated apoptosis in HCT-116 human colon adenocarcinoma cell line.

Among the eight phytochemicals (dihydrocarveol, sinapic acid, vanillic acid, ethylgallate, myrtenol, transcarveol, p-methoxycinnamic acid, and isoferulic acid) we tested, p-methoxycinnamic acid (p-MCA) [10 μM] showed the most potent in vitro growth inhibition on human colon adenocarcinoma (HCT-116 cells). Antiproliferative activity of p-MCA at 24h was associated with DNA damage, morphological changes and the results were comparable with doxorubicin. p-MCA induced phosphatidylserine translocation, increased the levels of reactive oxygen species (ROS), thiobarbituric acid reactive substances (TBARS), protein carbonyl content (PCC) and decreased enzymic antioxidant status (SOD, CAT, GPx) in HCT-116. p-MCA treatment increased the percentage of apoptotic cells, decreased the mitochondrial membrane potential and triggered cytochrome C release to cytosol. The induction of apoptosis by p-MCA was accompanied by an increase in caspase 3 and caspase 9 activities, increased expression of Bax and decreased expression of Bcl-2. Thus p-MCA induces mitochondria mediated intrinsic pathway of apoptosis in HCT-116 and has potential for treatment and prevention of colon cancer.

Potentiation of the activity of cisplatin in a human colon tumour xenograft model by auristatin PYE, a structural modification of dolastatin 10.

Dolastatin 10, a marine natural product peptide, is now known to act as a vascular disrupting agent (VDA). These VDA properties were not known when other aspects of its promising pre-clinical profile led to initial unsuccessful clinical trials. Auristatin PYE, a synthetic analogue of dolastatin 10, has demonstrated improved activity in preliminary in vivo studies. However, as with other VDAs, tumour eradication was incomplete due to the maintenance of functional vasculature supporting the viable tumour at the periphery of the tumour xenograft, meaning that once the VDA effect subsides, the tumour regrows. One possible strategy for removing this peripheral tumour involves combining VDA therapy with another anticancer drug with a different mechanism of action. Here, we evaluated the effect of combining auristatin PYE with cisplatin in an HCT-116 human colon adenocarcinoma xenograft model. The effects on the growth of subcutaneously implanted HCT-116 xenografts in mice following intraperitoneal administration of a single dose of 4 mgkg-1 cisplatin and intravenous administration of 1 mgkg-1 auristatin PYE were evaluated compared to the effect of each agent administered alone. The effects on the functional tumour vasculature were also assessed. Statistically significant potentiation (p<0.01) was noted with a 465% growth delay for the combination group compared to the control, and 142 and 310% growth delays for the cisplatin and auristatin PYE groups, respectively. Shut down of tumour vasculature in the combination group was similar to that observed with auristatin PYE on its own. Auristatin PYE demonstrated synergistic antitumour effects when combined with cisplatin, suggesting that a combination chemotherapy regimen would be the most effective strategy when applying this new anticancer drug.

A tryptophanol-derived oxazolopiperidone lactam is cytotoxic against tumors via inhibition of p53 interaction with murine double minute proteins

Inactivation of the p53 tumor suppressor protein by interaction with murine double minute (MDM) proteins, MDM2 and MDMX, is a common event in human tumors expressing wild-type p53. In these tumors, the simultaneous inhibition of these interactions with MDMs, for a full p53 reactivation, represents a promising anticancer strategy. Herein, we report the identification of a dual inhibitor of the p53 interaction with MDM2 and MDMX, the (S)-tryptophanol derivative OXAZ-1, from the screening of a small library of enantiopure tryptophanol-derived oxazolopiperidone lactams, using a yeast-based assay. With human colon adenocarcinoma HCT116 cell lines expressing wild-type p53 (HCT116 p53+/+) and its p53-null isogenic derivative (HCT116 p53−/−), it was shown that OXAZ-1 induced a p53-dependent tumor growth-inhibitory effect. In fact, OXAZ-1 induced p53 stabilization, up-regulated p53 transcription targets, such as MDM2, MDMX, p21, Puma and Bax, and led to PARP cleavage, in p53+/+, but not in p53−/−, HCT116 cells. In addition, similar tumor cytotoxic effects were observed for OXAZ-1 against MDMX-overexpressing breast adenocarcinoma MCF-7 tumor cells, commonly described as highly resistant to MDM2-only inhibitors. In HCT116 p53+/+ cells, the disruption of the p53 interaction with MDMs by OXAZ-1 was further confirmed by co-immunoprecipitation. It was also shown that OXAZ-1 potently triggered a p53-dependent mitochondria-mediated apoptosis, characterized by reactive oxygen species generation, mitochondrial membrane potential dissipation, Bax translocation to mitochondria, and cytochrome c release, and exhibited a p53-dependent synergistic effect with conventional chemotherapeutic drugs.Collectively, in this work, a novel selective activator of the p53 pathway is reported with promising antitumor properties to be explored either alone or combined with conventional chemotherapeutic drugs. Moreover, OXAZ-1 may represent a promising starting scaffold to search for new dual inhibitors of the p53–MDMs interaction.

Antiproliferative effect of phenylethanoid glycosides from Verbena officinalis L. on Colon Cancer Cell Lines

The cytotoxic effect of the aqueous extract from Verbena officinalis, was evaluated in vitro on DHD/K12/PROb rat colonic epithelial cell line and HCT-116 human colon adenocarcinoma cell line. In both cell lines, the IC50 values were lower than 20 μg/mL after 72 h of treatment. Bioassay guided fractionation led to the isolation of 12 phenylethanoid glycosides with anti-proliferative activity, five of them are being reported for the first time. The new compounds were elucidated as 4‴-acetyl-O-isoverbascoside, 2″,4″-diacetyl-O-verbascoside, 3‴,4‴-diacetyl-O-isoverbascoside, 4‴,6″-diacetyl-O-betonyoside A and 3‴,4‴-diacetyl-O-betonyoside A. The IC50 results suggest that antiproliferative activity is determined by not only the number of acetyl-groups but also their position in the aliphatic rings. Compounds exhibiting vicinal acetyl-groups in the sugar rings such as 3‴,4‴-diacetyl-O-isoverbascoside and 3‴,4‴-diacetyl-O-betonyoside A are particularly strong cytotoxic compounds against both cell lines. This investigation indicated that diacetyl-phenylethanoids might be valuable as cancer chemopreventive agents.

Oxazoloisoindolinones with in vitro antitumor activity selectively activate a p53-pathway through potential inhibition of the p53–MDM2 interaction

One of the most appealing targets for anticancer treatment is the p53 tumor suppressor protein. In half of human cancers, this protein is inactivated due to endogenous negative regulators such as MDM2. Actually, restoring the p53 activity, particularly through the inhibition of its interaction with MDM2, is considered a valuable therapeutic strategy against cancers with a wild-type p53 status. In this work, we report the synthesis of nine enantiopure phenylalaninol-derived oxazolopyrrolidone lactams and the evaluation of their biological effects as p53–MDM2 interaction inhibitors. Using a yeast-based screening assay, two oxazoloisoindolinones, compounds 1b and 3a, were identified as potential p53–MDM2 interaction inhibitors. The molecular mechanism of oxazoloisoindolinone 3a was further validated in human colon adenocarcinoma HCT116 cells with wild-type p53 (HCT116 p53+/+) and in its isogenic derivative without p53 (HCT116 p53−/−). Indeed, using these cells, we demonstrated that oxazoloisoindolinone 3a exhibited a p53-dependent in vitro antitumor activity through induction of G0/G1-phase cell cycle arrest and apoptosis. The selective activation of a p53-apoptotic pathway by oxazoloisoindolinone 3a was further supported by the occurrence of PARP cleavage only in p53-expressing HCT116 cells. Moreover, oxazoloisoindolinone 3a led to p53 protein stabilization and to the up-regulation of p53 transcriptional activity with increased expression levels of several p53 target genes, as p21(WAF1/CIP1), MDM2, BAX and PUMA, in p53+/+ but not in p53−/− HCT116 cells. Additionally, the ability of oxazoloisoindolinone 3a to block the p53–MDM2 interaction in HCT116 p53+/+ cells was confirmed by co-immunoprecipitation. Finally, the molecular docking analysis of the interactions between the synthesized compounds and MDM2 revealed that oxazoloisoindolinone 3a binds to MDM2. Altogether, this work adds, for the first time, the oxazoloisoindolinone scaffold to the list of chemotypes activators of a wild-type p53-pathway with promising antitumor activity. Moreover, it may open the way to the development of a new class of p53–MDM2 interaction inhibitors.