Cyclic gomesin as a scaffold for stabilising anticancer peptides and targeting melanoma cells

We previously presented data on the anticancer activity of a small library of synthetic flavonoid derivatives against glioblastoma and breast cancer lines in addition to several other human cancer cell lines. This data suggested that the chalcone structural backbone afforded more potent anticancer activity compared to its flavanone counterpart. Using this data, we selected three lead compounds: RK6, RK7, and RK15, to screen for activity against non‐cancerous cells to test for selectivity. Here we present updated IC50 values of these three analogs against MCF7 breast adenocarcinoma cells in addition to the previously reported data on the A‐172 glioblastoma cell line. To determine the selectivity of these lead compounds, RK6, RK7, and RK15 were screened against two non‐cancerous human cell lines: normal human astrocytes (NHA) and normal human mammary epithelial cells (NHME).The activity of our lead compounds against A‐172 and MCF7 cells were assessed using the XTT cell viability assay to determine respective IC50 values. As reported previously, the IC50 values of our lead compounds in the A‐172 cell line were 20 μM, 22 μM, and 3 μM for RK6, RK7, and RK15 respectively. Updated IC50 values of the compounds in the MCF7 cell line were 19 μM, 99 μM, and 12 μM for RK6, RK7, and RK15.In order to evaluate the selectivity of the lead compounds for cancerous vs. non‐cancerous cells, we utilized the Vialight cell viability assay to screen the chalcones in NHA and NHME cells. In the NHA cells, the determined IC50 values of RK6, RK7, and RK15 were 47 μM, 52 μM, and >200 μM respectively. IC50 values were 60 μM, >200 μM, and >200 μM for the NHME cells treated with RK6, RK7, and RK15 respectively. This data was used to calculate selectivity indices for our lead compounds against A‐172 cells compared to NHA cells and MCF7 cells compared to NHME cells. For RK6, there was a 2.3 fold selectivity for A‐172 cells and a 3.1 fold selectivity for MCF7 cells compared to their non‐cancerous comparator cells. The selectivity index for RK7 showed a 2.3 fold selectivity for A‐172 compared to the NHA. In these instances, RK6 and RK7 appear to be somewhat selective for cancerous vs. non‐cancerous cell lines. Given the IC50 values >200 μM for RK7 in NHA cells and RK15 in NHA and NHME cells, exact selectivity indices could not be determined. However, in these occurrences, it would appear that the compounds demonstrated a high degree of selectivity for cancerous vs. non‐cancerous cells.Collectively, these results suggest that our lead chalcone compounds are at least somewhat selective for cancerous vs. non‐cancerous cells. Interestingly, the most potent compound in the A‐172 and MCF7 cells, RK15, demonstrated the highest degree of selectivity. This is intriguing given that recent trends in anticancer drug discovery favor compounds that are selective for cancer cells. These findings may provide some insights into the yet to be elucidated mechanisms of cell death by these novel chalcone derivatives, and will form the basis of future studies.Support or Funding InformationThis work was supported in part by an AFPE Gateway Scholarship for the first author.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Selective Cancer Targeting via Aberrant Behavior of Cancer Cell-associated Glucocorticoid Receptor

Purpose The study aimed to disclose the anti-cancer activity of Pluchea indica tea leaves by evaluating the cytotoxicity on breast and cervical cancer cells, compared with non-cancer cells. Design/methodology/approach Two P. indica extracts were prepared using two solvents, namely hot water (PA) and ethanol (PE). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and clonogenic assays were applied to determine cytotoxic effect of both extracts toward cancer cells from human breast (MDA-MB-231 and MCF7) and cervix (SiHa, HeLa and C-33A) and also non-cancer Vero cells. Dichlorofluorescein diacetate (DCFDA)-staining assay was used to quantify the intracellular level of the reactive oxygen species (ROS). Correlation between the quantity of compounds present and the cytotoxicity of the extracts was analyzed by Pearson's method and a possible class of bioactive compounds was proposed based on the highest correlation coefficient (r). Findings Significant reduction in cell viability and proliferation capability was observed in all cancer cells after treatment with either PA or PE extract albeit PE was more effective. Lower toxicity was detected in Vero cells, indicating the selectivity and safety of extracts. The intracellular ROS level was augmented in treated cancer cells which were inversely correlated to cell viability, suggesting the cancer toxicity was likely induced by intracellular oxidative stress. As flavonoids were found abundantly in the extracts and flavonoids' content was the most related to the activity (r = 0.815), it was hypothesized that the flavonoids might play crucial roles in cancer cytotoxicity. Originality/value P. indica tea-leaf extracts can be a good source of promising anti-cancer agents with reduced side effects for breast and cervical cancer treatment.

The challenges translating glioblastoma therapies lie in safeguarding normal neuronal and glial tissue. Considering that the central nervous system has the lowest regenerative capacity, the exclusive focus on tumor eradication while neglecting normal tissue response, impedes proper healing. This oversight leads to toxicities ultimately culminating in patient demise with marginal gain in survival. BPM31510 is a nanodispersion encapsulating highly hydrophobic oxidized Coenzyme Q10 (CoQ10), allowing for delivery of supraphysiological CoQ10 concentrations into cancer cells. Initial investigations demonstrated differential responsiveness between cancer and non-cancer cells in vitro, an effect associated with high superoxide levels exclusively in cancer cells. To evaluate this divergence between cancer and non-cancer cells, a competitive co-culture cell-based assay was developed with labeled pairs of glioblastoma and non-cancer cells incubated up to three weeks with various concentrations of BPM31510 to ascertain optimal concentration leading to preferential growth of non-cancer cells. Human astrocyte (HA), mouse fibroblast (NIH 3T3), and rat astrocyte (TNC1) cells were paired with U251 human glioma, mouse GL261 and rat C6 glioma, respectively. Cell growth and spatial extent of the plate containing non-cancer or tumor cells were monitored every other day. Using doxorubicin as control, no dosage was discerned in which non-cancer cells exhibited advantage over tumor cells (i.e., either cultures became all tumor cells or both cell lines were eradicated). Using various concentrations of BPM31510, we identified an optimal dose in which cancer cells diminished while non-cancer cells predominated, reflected by markedly elevated mitochondrial superoxide levels in cancer cells prior to cell death. A co-culture cell-based assay was developed to identify an optimal dose of BPM31510 with selective preservation of non-cancer cells alongside tumor cell death. This approach elucidates developing a “fine tuning” strategy, wherein BPM31510 is titrated over time, thereby promoting normal tissue and healing, and ultimately leading to superior and prolonged functional outcomes.

Cyclic peptide therapeutics fill the gap between small molecules (<500 Da) and biologics (>5000 Da) as a separate class of drugs, combining the advantages of both in terms of high selectivity, bioavailability, synthetic accessibility and low toxicity. However, their conformational flexibility and proteolytic instability results in fast metabolism. Therefore, modification of their chemical and structural properties at the proteolytically vulnerable sites can circumvent these deficiencies. Several strategies exist to stabilise peptides through local or global constraints, side chain functionalisation or amide bond surrogates. This thesis describes the development and application of two chemical transformations to peptides with the aim of greatly enhancing their metabolic stability either via side chain to side chain cyclisation or disulfide modification. Following the functionalisation, a comparative evaluation of the pharmacological properties of the modified peptides was conducted with respect to the parent peptides. The first cyclisation strategy aimed to apply the redox-neutral chemistry developed in the burgeoning field of borrowing hydrogen technology to peptides, as a new class of tolerable substrates. These would contain the requisite amine and alcohol functional groups enabling their direct coupling to form the lactam or N-alkylated cycle. Numerous investigations into this transformation with a plethora of substrates and catalysts did not lead to the desired cyclisation. For the second approach, a simple one-pot procedure for the 'stapling' of disulfide bonds in native peptides was developed. Tris(2-carboxyethyl)phosphine mediated reduction of the disulfide bond was followed by concomitant insertion of a 'doubly electrophilic' carbon bridge to form a physiologically stable dithioacetal bond. The reaction was performed under mild, biocompatible reaction conditions, allowing for the facile conversion of native peptides into more stable analogues. The protocol was applicable to a range of bioactive peptides with a multitude of reactive functional groups demonstrating the high versatility of this approach. The importance and utility of the modified analogues were verified by testing their binding affinity and biological activity against the corresponding parent peptides. The impact of the disulfide modification on these properties for each peptide were analysed on a case-by-case basis. The SCS modified analogues of Oxytocin and Vasopressin maintained binding affinities in the same order of magnitude as their respective parent compounds across all corresponding receptors. Nevertheless, this was not the case for SCS-Octreotide and SCS-Somatostatin. Those peptides which maintained comparable binding affinities were then tested for their functional activity at the receptors of biological interest. SCS-Oxytocin retained its agonist potency displaying sub-nanomolar activity at the Oxytocin receptor, whilst SCS-Vasopressin exhibited sub micromolar functional response at the Vasopressin 1A receptor. Then the stability of the modified peptides in human serum was evaluated. In some cases, the enhancement in serum stability was vastly increased. In particular our modified SCS-Oxytocin analogue clearly demonstrated a significant improvement in serum half-life, as well as heat and pH stability over its native parent form. This disulfide stapling method could have wide-reaching application for the development of more stable therapeutic analogues.

Selenium chemoprevention by apoptosis has been well studied, but it is not clear whether selenium can activate early barriers of tumorigenesis, namely senescence and DNA damage response. To test this hypothesis, we treated normal and cancerous cells with a gradient concentration of sodium selenite, methylseleninic acid and methylselenocysteine for 48 h, followed by a recovery of 1-7 days. Here we show that selenium compounds at doses of </=LD(50) can induce cellular senescence, as evidenced by the expression of senescence-associated beta-galactosidase and 5-bromo-2-deoxyuridine incorporation, in normal but not cancerous cells. In response to clastogens, the ataxia telangiectasia mutated (ATM) protein is rapidly activated, which in turn initiates a cascade of DNA damage response. We found that the ATM pathway is activated by the selenium compounds, and the kinase activity is required for the selenium-induced senescence response. Pretreatment of the MRC-5 non-cancerous cells with the antioxidant N-acetylcysteine or 2,2,6,6-tetramethylpiperidine-1-oxyl suppresses the selenium-induced ATM activation and senescence. Taken together, the results suggest a novel role of selenium in the activation of early tumorigenesis barriers specific in non-cancerous cells, whereby selenium induces an ATM-dependent senescence response that depends on reactive oxygen species.

Biosensor device for the photo-specific detection of immuno-captured bladder cancer cells using hexaminolevulinate: An ex-vivo study.

Many quinoline-based compounds possess pharmacological properties that are beneficial for the control of various human diseases, including microbial infections, inflammatory-related disorders, cardiovascular conditions, and cancer. The anticancer activities of quinoline compounds are often rendered by their property of inhibiting protein kinases, proteasome activity, tubulin polymerization/depolymerization and DNA repair. To further improve the efficacy and specificity, we created and characterized several quinoline derivatives, which led us to identify CTRI-17 and CTRI-20 as promising leads. We examined their anti-proliferative properties using three human breast cancer cell lines (MDA-MB-468, MDA-MB-231 and MCF-7) and two matching non-cancer breast cell lines (184B5 and MCF10A). In addition, we also examined their efficacy using one leukemic cell line (K-562) and one cervical cell line (HeLa). Data from this study showed that the IC50 values of CTRI-17 and CTRI-20 were 0.1-0.3 μM and 1.2-2.4 μM ranges on cancer and non-cancer cell lines, respectively. Thus, both CTRI-17 and CTRI-20 killed cancer cells 10-20 times more effectively than non-cancer cells. This differential cell killing effects on cancer and non-cancer cells is a highly desirable property for potential anticancer therapeutics. We further found that cancer cells treated with the two compounds resulted in increases of pro-apoptotic proteins such as p53 and Bax, and decreases of anti-apoptotic proteins such as Bcl-2 and survivin. Our data obtained from flow cytometry and confocal microscopy showed that CTRI-17 and CTRI-20 induced prolonged cell cycle arrest at G2/M phase prior to causing apoptosis in cancer cells. However, the two compounds neither induced substantial cell cycle arrest nor caused high levels of apoptosis in non-cancer cells at the same drug concentration. We are currently in the process of determining the detailed functional mechanisms of CTRI-17 and CTRI-20. Citation Format: Indeewari K. Lindamulage, Hai-Yen Vu, Yi-Fang Lee, Hoyun Lee, Piyush Trivedi. Characterization of two novel quinoline derivatives that induce apoptosis in a cancer-specific manner. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 701. doi:10.1158/1538-7445.AM2013-701

In this study, we synthesized empty core-shell structured nanocapsules of Pluronic F127 and chitosan and characterized the thermal responsiveness of the nanocapsules in size and wall-permeability. Moreover, we determined the feasibility of using the nanocapsules to encapsulate small molecules for temperature-controlled release and intracellular delivery. The nanocapsules are ∼37 nm at 37 °C and expand to ∼240 nm when cooled to 4 °C in aqueous solutions, exhibiting >200 times change in volume. Moreover, the permeability of the nanocapsule wall is high at 4 °C (when the nanocapsules are swollen), allowing free diffusion of small molecules (ethidium bromide, MW = 394.3 Da) across the wall, while at 37 °C (when the nanocapsules are swollen), the wall-permeability is so low that the small molecules can be effectively withheld in the nanocapsule for hours. As a result of their thermal responsiveness in size and wall-permeability, the nanocapsules are capable of encapsulating the small molecules for temperature-controlled release and intracellular delivery into the cytosol of both cancerous (MCF-7) and noncancerous (C3H10T1/2) mammalian cells. The cancerous cells were found to take up the nanocapsules much faster than the noncancerous cells during 45 min incubation at 37 °C. Moreover, toxicity of the nanocapsules as a delivery vehicle was found to be negligible. The Pluronic F127-chitosan nanocapsules should be very useful for encapsulating small therapeutic agents to treat diseases particularly when it is combined with cryotherapy where the process of cooling and heating between 37 °C and hypothermic temperatures is naturally done.

Safe and targeted anticancer efficacy of a novel class of antioxidant-conjugated difluorodiarylidenyl piperidones: Differential cytotoxicity in healthy and cancer cells

A diagnosis of advanced ovarian cancer is the beginning of a long and arduous journey for a patient. Worldwide, approximately half of the individuals undergoing therapy for advanced cancer will succumb to the disease, or consequences of treatment. Well-known and widely-used chemotherapeutic agents such as cisplatin, paclitaxel, 5-fluorouracil, and doxorubicin are toxic to both cancer and non-cancerous cells, and have debilitating side effects Therefore, development of new targeted anticancer therapies that can selectively kill cancer cells while sparing the surrounding healthy tissues is essential to develop more effective therapies. We have developed a new class of synthetic curcumin analogs, diarylidenyl-piperidones (DAPs), which have higher anticancer activity and enhanced bio-absorption than curcumin. The DAP backbone structure exhibits cytotoxic (anticancer) activity, whereas the N-hydroxypyrroline (-NOH) moiety found on some variants functions as a cellular- or tissue-specific modulator (antioxidant) of cytotoxicity. The anticancer activity of the DAPs has been evaluated using a number of ovarian cancer cell lines, and the safety has been evaluated in a number of non-cancerous cell lines. Both variations of the DAP compounds showed similar levels of cell death in ovarian cancer cells, however the compounds with the -NOH modification were less toxic to non-cancerous cells. The selective cytotoxicity of the DAP–NOH compounds suggests that they will be useful as safe and effective anticancer agents. This article reviews some of the key findings of our work with the DAP compounds, and compares this to some of the targeted therapies currently used in ovarian cancer therapy.

The insulin-like growth factor (IGF) pathway represents one of the most studied molecular regulatory networks in oncology. Clinical trials investigating the therapeutic value of anti-IGF1 receptor (IGF1R) therapies in cancer, including prostate cancer, are ongoing. However, the multiple functions of the IGF network in the prostate are not entirely known. To elucidate the effects of IGF and insulin (INS) on prostate cells, we stimulated prostate cancer (PC3, DU145, LNCaP, DUCaP) and noncancerous prostate cells (EP156T, RWPE-1) and observed differing responses: whereas cancer cells responded to IGF and INS exposure by way of enhanced cell proliferation and glucose consumption, basal to luminal differentiation was induced in noncancerous cells. The same diverse responses were observed when the growth factor receptors IGF1R or INSR were overexpressed. Down-regulation of IGF1R or INSR isoform A (INSRA) also inhibited only proliferation of cancer cells. The proliferative response induced by the INSR in cancer cells was mediated solely by the INSRA. Moreover we observed that the receptors of the IGF network mutually influence their expression and exert redundant functions, thus underscoring the functional molecular network formed by IGF, INS, IGF1R, and INSR. Collectively we found that both IGF1R and INSRA have oncogenic effects in prostate cancer, but the IGF network also has important physiological functions in the noncancerous prostate. These data provide new insights into the biology of the IGF network in the prostate, thereby facilitating the design and interpretation of clinical studies investigating IGF1R targeting agents.

Methylselenol is hypothesized to be a critical selenium metabolite for anticancer action, and differential chemopreventive effects of methylselenol on cancerous and noncancerous cells may play an important role. In this study, the submicromolar concentrations of methylselenol were generated by incubating methionase with seleno-L methionine, and colon-cancer-derived HCT-116 cells and noncancerous colon NCM460 cells were exposed to methylselenol. Methylselenol exposure inhibited cell growth and led to an increase in G1 and G2 fractions with a concomitant drop in S-phase and an induction of apoptosis in HCT116, but to a much lesser extent in NCM460 colon cells. Similarly, the examination of mitogen-activated protein kinase (MAPK) and cellular myelocytomatosis oncogene (c-Myc) signaling status revealed that methylselenol inhibited the phosphorylation of extracellular-regulated kinase1/2 and p38 mitogen-activated protein kinase and the expression of c-Myc in HCT116 cells, but also to a lesser extent in NCM460 cells. The other finding is that methylselenol inhibits sarcoma kinase phosphorylation in HCT116 cells. In contrast, methylselenol upregulated the phosphorylation of sarcoma and focal adhesion kinase survival signals in the noncancerous NCM460 cells. Collectively, methylselenol's stronger potential of inhibiting cell proliferation/survival signals in the cancerous HCT116 cells when compared with that in noncancerous NCM460 cells may partly explain the potential of methylselenol's anticancer action.

Aloe vera gel extract: Safety evaluation for acute and chronic oral administration in Sprague-Dawley rats and anticancer activity in breast and lung cancer cells

Newpalladium (Pd)II and platinum (Pt)II complexes (C1–C5) from the Schiff base ligands, R-(phenyl)methanamine(L1), R-(pyridin-2-yl)methanamine (L2),and R-(furan-2-yl)methanamine (L3) (R-(E)-N-((1H-pyrrol-2-yl) methylene))are herein reported. The complexes (C1–C5) were characterized by FTIR, 1H and 13C NMR,UV–vis, and microanalyses. Single-crystal X-ray crystallographicanalysis was performed for the two ligands (L1–L2) and a Pt complex. Both L1 and L2 belong to P21/n monoclinicand P-1 triclinic space systems, respectively. The complex C5 belongs to the P21/c monoclinicspace group. The investigated molar conductivity of the complexesin DMSO gave the range 4.0–8.8 μS/cm, suggesting neutrality,with log P values ≥ 1.2692 ± 0.004, suggestinglipophilicity. The anticancer activity and mechanism of the complexeswere investigated against various human cancerous (Caco-2, HeLa, HepG2,MCF-7, and PC-3) and noncancerous (MCF-12A) cell lines using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) and Apopercentage assays, respectively. C5 demonstrated strong DNA-binding affinity for calf thymus DNA (CT-DNA)with a binding constant of 8.049 × 104 M–1. C3 reduced cell viability of all the six cell lines,which included five cancerous cell lines, by more than 80%. The C5 complex also demonstrated remarkably high selectivity withno cytotoxic activity toward the noncancerous breast cell line butreduced the viability of the five cancerous cell lines, which includedone breast cancer cell line, by more than 60%. Further studies arerequired to evaluate the selective toxicity of these two complexesand to fully understand their mechanism of action.