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Developing effective treatments for colorectal cancers through combinations of small-molecule approaches and immunotherapies present intriguing possibilities for managing these otherwise intractable cancers. During a broad-based, screening effort against multiple colorectal cancer cell lines, we identified indole-substituted quinolines (ISQ), such as N7,N7-dimethyl-3-(1-methyl-1H-indol-3-yl)quinoline-2,7-diamine (ISQ-1), as potent in vitro inhibitors of several cancer cell lines. We found that ISQ-1 inhibited Wnt signaling, a main driver in the pathway governing colorectal cancer development, and ISQ-1 also activated adenosine monophosphate kinase (AMPK), a cellular energy–homeostasis master regulator. We explored the effect of ISQs on cell metabolism. Seahorse assays measuring oxygen consumption rate (OCR) indicated that ISQ-1 inhibited complex I (i.e., NADH ubiquinone oxidoreductase) in the mitochondrial, electron transport chain (ETC). In addition, ISQ-1 treatment showed remarkable synergistic depletion of oncogenic c-Myc protein level in vitro and induced strong tumor remission in vivo when administered together with BI2536, a polo-like kinase-1 (Plk1) inhibitor. These studies point toward the potential value of dual drug therapies targeting the ETC and Plk-1 for the treatment of c-Myc–driven cancers.

An efficient method for regioselective synthesis of C-7 Mannich bases of 6-hydroxyaurones was accomplished by the N,N-dialkylaminomethylation using aminals prepared from dimethylamine, dipropylamine, bis(2-methoxyethyl)amine, N-methylbutylamine, N-methylbenzylamine, morpholine, piperidine, and 1-methylpiperazine. Further transformation of 7-(N,N-dialkylamino)methyl group in these aurones led to formation of C-7 acetoxymethyl and methoxymethyl derivatives of 6-hydroxyaurones, some of which showed promising inhibition of PC-3 prostate cancer cell proliferation in the high nanomolar to low micromolar range that exceeded that of cisplatin. Compound 12c (R3 = Ac, Ar = 3,4-OMePh) displays 75% inhibition of PC-3 prostate cancer cells proliferation at 300 nM concentration.

Structure-activity relationships (SAR) in the aurone pharmacophore identified heterocyclic variants of the (Z)-2-benzylidene-6-hydroxybenzofuran-3(2H)-one scaffold that possessed low nanomolar in vitro potency in cell proliferation assays using various cancer cell lines, in vivo potency in prostate cancer PC-3 xenograft and zebrafish models, selectivity for the colchicine-binding site on tubulin, and absence of appreciable toxicity. Among the leading, biologically active analogs were (Z)-2-((2-((1-ethyl-5-methoxy-1H-indol-3-yl)methylene)-3-oxo-2,3-dihydrobenzofuran-6-yl)oxy)acetonitrile (5a) and (Z)-6-((2,6-dichlorobenzyl)oxy)-2-(pyridin-4-ylmethylene)benzofuran-3(2H)-one (5b) that inhibited in vitro PC-3 prostate cancer cell proliferation with IC50 values below 100 nM. A xenograft study in nude mice using 10 mg/kg of 5a had no effect on mice weight, and aurone 5a did not inhibit, as desired, the human ether-à-go-go-related (hERG) potassium channel. Cell cycle arrest data, comparisons of the inhibition of cancer cell proliferation by aurones and known antineoplastic agents, and in vitro inhibition of tubulin polymerization indicated that aurone 5a disrupted tubulin dynamics. Based on molecular docking and confirmed by liquid chromatography-electrospray ionization-tandem mass spectrometry studies, aurone 5a targets the colchicine-binding site on tubulin. In addition to solid tumors, aurones 5a and 5b strongly inhibited in vitro a panel of human leukemia cancer cell lines and the in vivo myc-induced T cell acute lymphoblastic leukemia (T-ALL) in a zebrafish model.

Mitochondria are ideal anti-tumor target due to mitochondria's central regulation role in cell apoptosis and tumor resistance to apoptosis. There are several challenges for mitochondrial targeting drug delivery, including complex multistep preparations, low drug- loading and systemic toxicity from the carriers. To address these issues, we firstly constructed mitochondria-targeting nanodrugs self-assembled from 9-O-octadecyl substituted berberine derivative (BD) using simple nano-precipitation approach. BD-based nanodrugs were modified by DSPE-PEG2000 (distearylphosphatidylethanolamine- methoxypolyethylene glycol 2000) to increase stability. Negatively charged hyaluronic acid (HA) was further coated to conceal positive charges and achieve tumor targeting. PEG and HA dually modified BD NDs (HA/PEG/BD NDs) were prepared with surface charge of −25.8 mV and high drug loading >70%. The degradation of HA by hyaluronidase (HAase) at tumor tissue allowed the exposure of the positively charged PEG/BD NDs to the cells, which is beneficial for cell uptake and further lysosome escape and mitochondrial targeting. Then, HA/PEG/BD NDs were investigated to induce apoptosis through dissipating mitochondria membrane potential, releasing cytochrome C, increasing the activities of caspase 9/3, activating the pro-apoptotic Bax, suppressing the anti-apoptotic Bcl-2 and upregulating ROS levels. In the A549 xenografted tumor model, HA/PEG/BD NDs exhibited obvious tumor cell mitochondrial targeting and significant anti-tumor efficacy. Overall, comparing to conventional nanoparticles, mitochondrial targeting HA/PEG/BD NDs provide a new strategy for cancer treatment with enhanced drug-loading, relatively simplified preparation processes and reduced carrier toxicities.

The importance of upregulated Wnt signaling in colorectal cancers led to efforts to develop inhibitors that target β-catenin in this pathway. We now report that several “Wnt inhibitors” that allegedly target β-catenin actually function as mitochondrial proton uncouplers that independently activate AMPK and concomitantly inhibit Wnt signaling. As expected for a process in which mitochondrial uncoupling diminishes ATP production, a mitochondrial proton uncoupler, FCCP, and a glucose metabolic inhibitor, 2-DG, activated AMPK and inhibited Wnt signaling. Also consistent with these findings, a well-known “Wnt inhibitor”, FH535, functioned as a proton uncoupler, and in support of this finding, the N-methylated analog, 2,5-dichloro-N-methyl-N-(2-methyl-4-nitrophenyl)benzenesulfonamide (FH535-M) was inactive as an uncoupler and Wnt inhibitor. Apart from suggesting an opportunity to develop dual Wnt inhibitors and AMPK activators, these findings provide a cautionary tale that claims for Wnt inhibition alone require scrutiny as possible mitochondrial proton uncouplers or inhibitors of the electron transport chain.

Fluorinated, phenylethynyl-substituted heterocycles that possessed either an N-methylamino or N,N-dimethylamino group attached to heterocycles including pyridines, indoles, 1H-indazoles, quinolines, and isoquinolines inhibited the proliferation of LS174T colon cancer cells in which the inhibition of cyclin D1 and induction of the cyclin-dependent kinase inhibitor-1 (i.e., p21Wif1/Cip1) served as a readout for antineoplastic activity at a cellular level. On a molecular level, these agents, particularly 4-((2,6-difluorophenyl)ethynyl)-N-methylisoquinolin-1-amine and 4-((2,6-difluorophenyl)ethynyl)-N,N-dimethylisoquinolin-1-amine, bound and inhibited the catalytic subunit of methionine S-adenosyltransferase-2 (MAT2A).

The factors affecting intracellular ice formation (IIF) and growth is essential to the mechanistic understanding of cellular damage through freezing. In the aid of high speed and high-resolution cryo-imaging technology, the broad bean intracellular ice formation and growth processes were successfully captured during freezing. Cytochalasin B（CB）was used to solubilize the cytoskeleton. Images of IIF were compared between cells with and without cytoskeleton. The behavior of intracellular ice crystal formation in plant tissues with or without CB was evaluated using changes of cell areas, the probability of crystallization, and growing rate of intracellular ice crystal. Moreover, light intensity figures were used to determine cell damage. This study showed that the cytoskeleton was involved in ice crystal nucleation mechanism during freezing responses of the plant cells.

Cytisine-linked isoflavonoids (CLIFs) inhibited PC-3 prostate and LS174T colon cancer cell proliferation by inhibiting a peroxisomal bifunctional enzyme. A pull-down assay using a biologically active, biotin-modified CLIF identified the target of these agents as the bifunctional, peroxisomal enzyme, hydroxysteroid 17β-dehydrogenase-4 (HSD17B4). Additional studies with truncated versions of HSD17B4 established that CLIFs specifically bind the C-terminus of HSD17B4 and selectively inhibited the enoyl CoA hydratase but not the D-3-hydroxyacyl CoA dehydrogenase activity. HSD17B4 was overexpressed in prostate and colon cancer tissues, knocking down HSD17B4 inhibited cancer cell proliferation, suggesting that HSD17B4 is a potential biomarker and drug target and that CLIFs are potential probes or therapeutic agents for these cancers.

Summary Aberrant activation of Wnt signaling triggered by mutations in either Adenomatous Polyposis Coli (APC) or CTNNB1 (β-catenin) is a hallmark of colorectal cancers (CRC). As part of a program to develop epigenetic regulators for cancer therapy, we developed carboxamide-substituted benzhydryl amines (CBAs) bearing either aryl or heteroaryl groups that selectively targeted histone lysine demethylases (KDMs) and functioned as inhibitors of the Wnt pathway. A biotinylated variant of N-((5-chloro-8-hydroxyquinolin-7-yl) (4-(diethylamino)phenyl)-methyl)butyramide (CBA-1) identified KDM3A as a binding partner. KDM3A is a Jumonji (JmjC) domain-containing demethylase that is significantly upregulated in CRC. KDM3A regulates the demethylation of histone H3's lysine 9 (H3K9Me2), a repressive marker for transcription. Inhibiting KDM3 increased H3K9Me2 levels, repressed Wnt target genes, and curtailed in vitro CRC cell proliferation. CBA-1 also exhibited in vivo inhibition of Wnt signaling in a zebrafish model without displaying in vivo toxicity., Graphical Abstract, Highlights • A class of carboxamide-substituted benzhydryl amine (CBA) Wnt inhibitors • A biological active, biotinylated CBA to identify KDM3A as a direct target • CBA-1 interacted with the Mn2+ ion in the JmjC domains of KDM3A/3B • CBA-1 inhibited Wnt signaling in colon cancer cells and in zebrafish models, Biological Sciences; Biochemistry; Biochemical Mechanism

Fluorinated phenylethynyl-substituted heterocycles inhibit cyclin D1, induce cyclin-dependent kinase inhibitor-1 and block the proliferation of colorectal cancer cells.

The toxicity and irritation associated with high amounts of surfactants restrict the extensive utilization of microemulsions. To address these shortcomings, employing mixed oils to enlarge microemulsion areas therefore reducing surfactant contents is a promising strategy. However, what kinds of mixed oils are more efficient in enlarging microemulsion areas still remains unclear. In this research, we found that the chain length and degree of unsaturation of oils play a key role in enlarging microemulsion areas. The combination of moderate chain saturated oil caprylic/capric triglyceride (GTCC) with long chain unsaturated oil glycerol trioleate significantly increased the microemulsion areas. Solubility of ibuprofen in the mixed oils was unexpectedly and remarkably increased (almost 300mg/mL) compared with that (around 100mg/mL) of the single oil (GTCC), which also resulted in greatly increased solubility of ibuprofen in mixed oils-containing microemulsions. By optimizing the mixed oil formulation, the absolute amount of surfactant in drug-loaded microemulsions was reduced but increased drug oral bioavailability in rats was maintained. It could be concluded that the combined use of moderate chain oils and long chain unsaturated oils could not only acquire enlarged microemulsion areas but also enhanced drug solubility, therefore doubly reducing surfactant amount, which is extremely beneficial for developing safe microemulsions.

So far, the components responsible for the neuroprotective effects of Calculus bovis are unclear. Cholesterol, one of the major components in Calculus bovis, is easily oxidized into oxysterols, which possess direct or indirect neuroprotective effects proved by our and others' previous studies. Therefore, a liquid chromatography with mass spectrometry method coupled with ultrasonic extraction and solid-phase extraction was developed for the determination of neuroprotective oxysterols in Calculus bovis, human gallstones, and traditional Chinese medicine preparations. Chromatographic separation was achieved on a C18 column with isocratic elution at a flow rate of 1 mL/min. The established method showed good linearity (R(2) > 0.998), sensitivity with low limits of detection (0.06-0.39 μg/g), acceptable precisions (relative standard deviations ≤ 7.4%), stability (relative standard deviations ≤ 5.9%), and satisfactory accuracy (92.4-102.9%) for all analytes identified by different retention times, which could be applied for the determination of oxysterols. Five kinds of oxysterols proved to function as neuroprotectants were detected at different concentrations. Among them, 7β-hydroxycholesterol and cholestane-3β,5α,6β-triol were rather abundant in the samples. It could be concluded that the potential neuroprotective components in Calculus bovis may be these oxysterols.

This research aims to synthesize lipophilic berberine derivatives and evaluate their antiglioma effects on C6 and U87 cells. The introduction of substituents with various carbon chain lengths on C-13- or C-9-O-position of the berberine scaffold led to the discovery of several potent inhibitors against glioblastoma cells. Derivatives substituted with the carbon chains of moderate length (twelve carbons) displayed improved lipophilicity and the strongest inhibitory effects. Several compounds presented dose-dependent repression against proliferation (IC50, 1.12–6.12 μM) and blocked migration and invasion by over 60% at lower dose levels. Furthermore, preliminary research about the underlying mechanism for the enhanced antiglioma ability indicated that these analogues preferentially localized into mitochondria, inducing the up-regulation of ROS production. Overall, these compounds represent promising candidates to combat glioblastoma and highlight new insight into the antiglioma therapy through interaction with mitochondria.

Sijia Zheng,1,* Yanqi Xie,1,* Yuan Li,1 Ling Li,1 Ning Tian,1 Wenbo Zhu,2 Guangmei Yan,2 Chuanbin Wu,1 Haiyan Hu1 1School of Pharmaceutical Sciences, 2Department of Pharmacology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, People's Republic of China *These authors contributed equally to this workAbstract: The objective of this research was to develop and evaluate high drug-loading ligand-modified nanomicelles to deliver a steroidal compound to the brain. YC1 (5α-cholestane-24-methylene-3β, 5α, 6β, 19-tetraol), with poor solubility and limited access to the brain, for the first time, has been proved to be an effective neuroprotective steroid by our previous studies. Based on the principle of ‘like dissolves like’, cholesterol, which shares the same steroidal parent nucleus with YC1, was selected to react with sodium alginate, producing amphiphilic sodium alginate–cholesterol derivatives (SACDs). To increase the grafting ratio and drug loading, cholesterol was converted to cholesteryl chloroformate, for the first time, before reacting with sodium alginate. Further, lactoferrin was conjugated on SACDs to provide lactoferrin-SACDs (Lf-SACD), which was established by immune electron microscopy (IEM) and self-assembled into brain-targeting nanomicelles. These nanomicelles were negatively charged and spherical in nature, with an average size of

In an effort to screen natural compounds and their derivatives as novel agents for cancer treatment, we developed two semisynthetic nature compounds, A14 and A52, which have potencies in an in vitro cell proliferation studies against LS174T and PC-3 cells in the mid-nanomolar range. A14 inhibited more than 90% of in vitro cancer cell proliferation around 300 nM and also inhibited in vivo PC-3 prostate cancer xenografts in nude mice. Cell morphology studies and comparisons with known antineoplastic agents suggested that A14 disrupted microtubule organization. In vitro tubulin polymerization assay suggested that A14 inhibited this process. Based on molecular docking, A14 fits very well in the colchicine-binding site at the interface between the alpha-tubulin and beta-tubulin. Further analyses suggest that A14 sensitivity may correlate with CDK/cyclin activity. A14 inhibited PC-3 prostate cancer xenografts in nude mice. In addition to solid tumors, A14 and its analog A52 strongly inhibited a panel of human leukemia cancer cell lines. Furthermore, these compounds inhibited Myc-induced T cell acute lymphoblastic leukemia (T-ALL) in a zebrafish model. Although many tubulin inhibitors were available, there is no compound that targeting the colchicine-binding site has been approved by FDA for cancer treatment. This new family of tubulin polymerization inhibitors could be further developed for the treatment of both solid and liquid tumors. Citation Format: Yanqi Xie, Liliia Kril, Wen Zhang, Mykhaylo Frasinyuk, Svitlana Bondarenko, Elizabeth Hausman, Zachary Martin, Przemyslaw Wyrebek, Tianxin Yu, Changguo Zhan, Vitaliy Sviripa, Jessica Blackburn, David Watt, Chunming Liu. Novel tubulin polymerization inhibitors repress tumor xenografts in nude mice and leukemia in zebrafish [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2674.