A hot and cold tumor­related prognostic signature for stage II colorectal cancer.

Globally, colorectal cancer (CRC) is one of the most lethal and prevalent malignancies. Based on the presence of immune cell infiltration in the tumor microenvironment, CRC can be divided into immunologically 'hot' or 'cold' tumors, which in turn leads to the differential efficacy of immunotherapy. However, the immune characteristics of hot and cold CRC tumors remain largely elusive, prompting further investigation of their properties regarding the tumor microenvironment. In the present study, a predictive model was developed based on the differential expression of proteins between cold and hot CRC tumors. First, the differentially expressed proteins (DEPs) were identified using digital spatial profiling and mass spectrometry-based proteomics analysis, and the pathway features of the DEPs were analyzed using functional enrichment analysis. A novel eight-gene signature prognostic risk model was developed (IDO1, MAT1A, NPEPL1, NT5C, PTGR2, RPL29, TMEM126A and TUBB4B), which was validated using data obtained from The Cancer Genome Atlas. The results revealed that the risk score of the eight-gene signature acted as an independent prognostic indicator in patients with stage II CRC (T3-4N0M0). It was also found that a high-risk score in the eight-gene signature was associated with high immune cell infiltration in patients with CRC. Taken together, these findings revealed some of the differential immune characteristics of hot and cold CRC tumors, and an eight-gene signature prognostic risk model was developed, which may serve as an independent prognostic indicator for patients with stage II CRC (T3-4N0M0).

tRF-20-M0NK5Y93-induced MALAT1 promotes colon cancer metastasis through alternative splicing of SMC1A.

Recent studies have indicated that changes in the tumor microenvironment, such as hypoxia, result in the discrepant expression of noncoding small RNA tRNA-derived fragments (tRFs), affecting the phenotype of tumor metastasis. The biological function of tRFs in tumors has attracted increasing attention, but the mechanism by which tRFs mediate tumor metastasis has not been clarified. The direct regulatory relationship between tRFs and lncRNAs and the mechanism by which noncoding RNAs regulate alternative splicing are still unknown. In this study, the mechanism of tRF-mediated SMC1A alternative splicing and regulation of colon cancer metastasis was studied from multiple dimensions of cell, molecule, animal and clinical. Our present studies revealed that tRF-20-M0NK5Y93 inhibits colon cancer metastasis and that there is a significant correlation between the expression of tRFs, metastasis-associated lung adenocarcinoma transcript 1 (MALAT-1), and SRSF2 through complete transcriptional sequencing and bioinformatics. Mechanistic investigations indicated that tRFs could regulate the expression of MALAT-1 by binding to specific sites on MALAT-1. MALAT1, which is a long noncoding RNA (lncRNA), regulates alternative splicing of (structural maintenance of chromosomes 1A) SMC1A by interaction with SRSF2, resulting in discrepant expression of various isoforms, SMC1A001, SMC1A201, SMC1A005, and SMC1A003. Our findings revealed the interaction between different types of noncoding RNAs on alternative splicing, which is expected to be a novel potential therapeutic target.

Synthesis and evaluation of stereoisomers of methylated catechin and epigallocatechin derivatives on modulating P-glycoprotein-mediated multidrug resistance in cancers.

P-glycoprotein (P-gp; ABCB1)-mediated drug efflux causes multidrug resistance in cancer. Previous synthetic methylated epigallocatechin (EGC) possessed promising P-gp modulating activity. In order to further improve the potency, we have synthesized some novel stereoisomers of methylated epigallocatechin (EGC) and gallocatechin (GC) as well as epicatechin (EC) and catechin (C). The (2R, 3S)-trans-methylated C derivative 25 and the (2R, 3R)-cis-methylated EC derivative 31, both containing dimethyoxylation at ring B, tri-methoxylation at ring D and oxycarbonylphenylcarbamoyl linker between ring D and C3, are the most potent in reversing P-gp mediated drug resistance with EC50 ranged from 32 nM to 93 nM. They are non-toxic to fibroblast with IC50 > 100 µM. They can inhibit the P-gp mediated drug efflux and restore the intracellular drug concentration to a cytotoxic level. They do not downregulate surface P-gp protein level to enhance drug retention. They are specific for P-gp with no or low modulating activity towards MRP1- or BCRP-mediated drug resistance. In summary, methylated C 25 and EC 31 derivatives represent a new class of potent, specific and non-toxic P-gp modulator.

Increased S1P induces S1PR2 internalization to blunt the sensitivity of colorectal cancer to 5-fluorouracil via promoting intracellular uracil generation.

Sphingosine-1-phosphate (S1P), the backbone of most sphingolipids, activating S1P receptors (S1PRs) and the downstream G protein signaling has been implicated in chemoresistance. In this study we investigated the role of S1PR2 internalization in 5-fluorouracil (5-FU) resistance in human colorectal cancer (CRC). Clinical data of randomly selected 60 CRC specimens showed the correlation between S1PR2 internalization and increased intracellular uracil (P < 0.001). Then we explored the regulatory mechanisms in CRC model of villin-S1PR2-/- mice and CRC cell lines. We showed that co-administration of S1P promoted S1PR2 internalization from plasma membrane (PM) to endoplasmic reticulum (ER), thus blunted 5-FU efficacy against colorectal tumors in WT mice, compared to that in S1PR2-/- mice. In HCT116 and HT-29 cells, application of S1P (10 µM) empowered S1PR2 to internalize from PM to ER, thus inducing 5-FU resistance, whereas the specific S1PR2 inhibitor JTE-013 (10 µM) effectively inhibited S1P-induced S1PR2 internalization. Using Mag-Fluo-AM-labeling [Ca2+]ER and LC-ESI-MS/MS, we revealed that internalized S1PR2 triggered elevating [Ca2+]ER levels to activate PERK-eLF2α-ATF4 signaling in HCT116 cells. The activated ATF4 upregulated RNASET2-mediated uracil generation, which impaired exogenous 5-FU uptake to blunt 5-FU therapy. Overall, this study reveals a previously unrecognized mechanism of 5-FU resistance resulted from S1PR2 internalization-upregulated uracil generation in colorectal cancer, and provides the novel insight into the significance of S1PR2 localization in predicting the benefit of CRC patients from 5-FU-based chemotherapy.

SphK2 confers 5-fluorouracil resistance to colorectal cancer via upregulating H3K56ac-mediated DPD expression.

Aberrant sphingolipid metabolism has been implicated in chemoresistance, but the underlying mechanisms are still poorly understood. Herein we revealed a previously unrecognized mechanism of 5-fluorouracil (5-FU) resistance contributed by high SphK2-upregulated dihydropyrimidine dehydrogenase (DPD) in colorectal cancer (CRC), which is evidenced from human CRC specimens, animal models, and cancer cell lines. TMA samples from randomly selected 60 CRC specimens firstly identified the clinical correlation between high SphK2 and increased DPD (p < 0.001). Then the regulatory mechanism was explored in CRC models of villin-SphK2 Tg mice, SphK2-/-mice, and human CRC cells xenografted nude mice. Assays of ChIP-Seq and luciferase reporter gene demonstrated that high SphK2 upregulated DPD through promoting the HDAC1-mediated H3K56ac, leading to the degradation of intracellular 5-FU into inactive α-fluoro-ß-alanine (FBAL). Lastly, inhibition of SphK2 by SLR080811 exhibited excellent inhibition on DPD expression and potently reversed 5-FU resistance in colorectal tumors of villin-SphK2 Tg mice. Overall, this study manifests that SphK2high conferred 5-FU resistance through upregulating tumoral DPD, which highlights the strategies of blocking SphK2 to overcome 5-FU resistance in CRC.

Novel α-Lipoic Acid/3-n-Butylphthalide Conjugate Enhances Protective Effects against Oxidative Stress and 6-OHDA Induced Neuronal Damage.

Neurodegenerative diseases are irreversible conditions that result in progressive degeneration and death of nerve cells. Although the underlying mechanisms may vary, oxidative stress is considered to be one of the major causes of neuronal loss. Importantly, there are still no comprehensive treatments to completely cure these diseases. Therefore, protecting neurons from oxidative damage may be the most effective therapeutic strategy. Here we report a neuroprotective effects of a novel hybrid compound (dlx-23), obtained by conjugating α-lipoic acid (ALA), a natural antioxidant agent, and 3-n-butylphthalide (NBP), a clinical anti-ischemic drug. Dlx-23 protected against neuronal death induced by both H2O2 induced oxidative stress in Cath.-a-differentiated (CAD) cells and 6-OHDA, a toxin model of Parkinson's disease (PD) in SH-SY5Y cells. These activities proved to be more potent than the parent compound (ALA) alone. Dlx-23 scavenged free radicals, increased glutathione levels, and prevented mitochondria damage. In addition, live imaging of primary cortical neurons demonstrated that dlx-23 protected against neuronal growth cone damage induced by H2O2. Taken together these results suggest that dlx-23 has substantial potential to be further developed into a novel neuroprotective agent against oxidative damage and toxin induced neurodegeneration.

S1PR2 inhibitors potently reverse 5-FU resistance by downregulating DPD expression in colorectal cancer.

In this study, S1PR2 was reckoned as a brand-new GPCR target for designing inhibitors to reverse 5-FU resistance. Herein a series of pyrrolidine pyrazoles as the S1PR2 inhibitors were designed, synthesized and evaluated for their activities of anti-FU-resistance. Among them, the most promising compound JTE-013, exhibited excellent inhibition on DPD expression and potent anti-FU-resistance activity in various human cancer cell lines, along with the in vivo HCT116DPD cells xenograft model, in which the inhibition rate of 5-FU was greatly increased from 13.01%-75.87%. The underlying mechanism was uncovered that JTE-013 demonstrated an anti-FU-resistance activity by blocking S1PR2 internalization to the endoplasmic reticulum (ER), which inhibited the degradation of 5-FU into α-fluoro-ß-alanine (FBAL) by downregulating tumoral DPD expression. Overall, JTE-013 could serve as the lead compound for the discovery of new anti-FU-resistance drugs. SIGNIFICANCE: This study provides novel insights that S1PR2 inhibitors could sensitize 5-FU therapy in colorectal cancer.

Structural modification of oridonin via DAST induced rearrangement.

A simple and efficient protocol was developed for the syntheses of oridonin analogues, i.e. 6,20-epoxy ent-kaurane diterpenoid analogues from oridonin via diethylaminosulfur trifluoride (DAST) promoted rearrangement, most of which exhibited superior anticancer activities compared with their precursor.

Extending the structure-activity relationship study of marine natural ningalin B analogues as P-glycoprotein inhibitors.

In the present study, a total of 25 novel ningalin B analogues were synthesized and evaluated for their P-gp modulating activity in a P-gp overexpressed breast cancer cell line LCC6MDR. Preliminary structure-activity study shows that A ring and its two methoxy groups are important pharmacophores for P-gp inhibiting activity. Among all derivatives, 23 is the most potent P-gp modulator with EC50 of 120-165 nM in reversing paclitaxel, DOX, vinblastine and vincristine resistance. It is relatively safe to use with selective index at least greater than 606 compared to verapamil. Mechanistic study demonstrates that compound 23 reverses P-gp mediated drug resistance by inhibiting transport activity of P-gp, thereby restoring intracellular drug accumulation. In summary, our study demonstrates that ningalin B analogue 23 is a non-cytotoxic and effective P-gp chemosensitizer that can be used in the future for reversing P-gp mediated clinical cancer drug resistance.

Synthesis, biological evaluation, and physicochemical property assessment of 4-substituted 2-phenylaminoquinazolines as Mer tyrosine kinase inhibitors.

Current results identified 4-substituted 2-phenylaminoquinazoline compounds as novel Mer tyrosine kinase (Mer TK) inhibitors with a new scaffold. Twenty-one 2,4-disubstituted quinazolines (series 4-7) were designed, synthesized, and evaluated against Mer TK and a panel of human tumor cell lines aimed at exploring new Mer TK inhibitors as novel potential antitumor agents. A new lead, 4b, was discovered with a good balance between high potency (IC50 0.68µM) in the Mer TK assay and antiproliferative activity against MV4-11 (GI50 8.54µM), as well as other human tumor cell lines (GI50<20µM), and a desirable druglike property profile with low logP value (2.54) and high aqueous solubility (95.6µg/mL). Molecular modeling elucidated an expected binding mode of 4b with Mer TK and necessary interactions between them, thus supporting the hypothesis that Mer TK might be a biologic target of this kind of new active compound.

Optimization of permethyl ningalin B analogs as P-glycoprotein inhibitors.

In the present study, a total of 9 novel permethyl ningalin B analogs have been synthesized and evaluated for their P-gp modulating activity in a P-gp overexpressed breast cancer cell line LCC6MDR. Among these derivatives, compound 12 with dimethoxy groups at rings A and B and tri-substitution at ring C with ortho-methoxyethylmorpholine, meta-bromo and para-benzyloxy groups displays the most potent P-gp modulating activity with EC50 of 423 nM to reverse paclitaxel resistance. It is non-toxic towards L929 fibroblast with IC50 greater than 100 µM and with selective index greater than 236. Its mechanism to reverse P-gp mediated drug resistance is by virtue of inhibiting transport activity of P-gp, restoring intracellular drug accumulation and eventually chemosensitizing the cancer cells to anticancer drug again. Moreover, compound 12 showed better solubility (405 ng/mL) than hit compound 1 in phosphate buffer (pH 4.0). In summary, our study demonstrates that permethyl ningalin B derivative 12 is non-toxic and efficient P-gp inhibitor that is a potential candidate to be used clinically to reverse P-gp mediated cancer drug resistance.

Optimization of N-aryl-6-methoxy-1,2,3,4-tetrahydroquinolines as tubulin polymerization inhibitors.

Thirteen new N-aryl 1,2,3,4-tetrahydroquinoline compounds (4a-f, 6a-c, and 8a-d) were synthesized and evaluated for antitumor activity and drug-like properties. Compound 4a exhibited high inhibitory potency with low nanomolar GI50 values of 16-20 nM in cellular assays, including excellent activity against the P-glycoprotein overexpressing cell line KBvin. Compound 4a inhibited colchicine binding to tubulin and tubulin assembly with an IC50 value of 0.85 µM, superior to the reference compound CA4 (1.2 µM) in the same assay. In addition, 4a also exhibited highly improved water solubility (75 µg/mL) and a suitable logP value (3.43) at pH 7.4. With a good balance between antitumor potency and drug-like properties, compound 4a could be a new potential drug candidate for further development. Current results on SAR studies and molecular modeling provided more insight about this class of compounds as tubulin polymerization inhibitors targeting the colchicine site.

Potent and Nontoxic Chemosensitizer of P-Glycoprotein-Mediated Multidrug Resistance in Cancer: Synthesis and Evaluation of Methylated Epigallocatechin, Gallocatechin, and Dihydromyricetin Derivatives.

We are interested in developing novel natural product-derived P-gp inhibitors to reverse cancer drug resistance. Here, we have synthesized 55 novel derivatives of methylated epigallocatechin (EGC), gallocatechin (GC), and dihydromyricetin (DHM). Three EGC derivatives (23, 35, and 36) and three GC derivatives (50, 51, and 53) are significantly better than epigallocatechin gallate (EGCG) with a relative fold (RF) ranging from 31.4 to 53.6. The effective concentration (EC50) of 23 and 51 ranges from 102 to 195 nM. Compounds 23 and 51 are noncytotoxic to fibroblasts with IC50 > 100 µM. Compound 23 is specific for P-gp without modulating activity toward MRP1 or BCRP. Compounds 23 and 51 are non-P-gp substrates. Important pharmacophores for P-gp modulation were identified. In summary, methylated EGC and GC derivatives represent a new class of potent, specific, noncytotoxic, and nonsubstrate P-gp modulators.

Modification of marine natural product ningalin B and SAR study lead to potent P-glycoprotein inhibitors.

In this study, new marine ningalin B analogues containing a piperazine or a benzoloxy group at ring C have been synthesized and evaluated on their P-gp modulating activity in human breast cancer and leukemia cell lines. Their structure-activity relationship was preliminarily studied. Compounds 19 and 20 are potent P-gp inhibitors. These two synthetic analogues of permethyl ningalin B may be potentially used as effective modulators of P-gp-mediated drug resistance in cancer cells.

Optimization of 4-(N-cycloamino)phenylquinazolines as a novel class of tubulin-polymerization inhibitors targeting the colchicine site.

The 6-methoxy-1,2,3,4-tetrahydroquinoline moiety in prior leads 2-chloro- and 2-methyl-4-(6-methoxy-3,4-dihydroquinolin-1(2H)-yl)quinazoline (1a and 1b) was modified to produce 4-(N-cycloamino)quinazolines (4a-c and 5a-m). The new compounds were evaluated in cytotoxicity and tubulin inhibition assays, resulting in the discovery of new tubulin-polymerization inhibitors. 7-Methoxy-4-(2-methylquinazolin-4-yl)-3,4-dihydroquinoxalin- 2(1H)-one (5f), the most potent compound, exhibited high in vitro cytotoxic activity (GI50 1.9-3.2 nM), significant potency against tubulin assembly (IC50 0.77 µM), and substantial inhibition of colchicine binding (99% at 5 µM). In mechanism studies, 5f caused cell arrest in G2/M phase, disrupted microtubule formation, and competed mostly at the colchicine site on tubulin. Compound 5f and N-methylated analogue 5g were evaluated in nude mouse MCF7 xenograft models to validate their antitumor activity. Compound 5g displayed significant in vivo activity (tumor inhibitory rate 51%) at a dose of 4 mg/kg without obvious toxicity, whereas 5f unexpectedly resulted in toxicity and death at the same dose.

Discovery of novel antitumor dibenzocyclooctatetraene derivatives and related biphenyls as potent inhibitors of NF-κB signaling pathway.

Several dibenzocyclooctatetraene derivatives (5-7) and related biphenyls (8-11) were designed, synthesized, and evaluated for inhibition of cancer cell growth and the NF-κB signaling pathway. Compound 5a, a dibenzocyclooctatetraene succinimide, was discovered as a potent inhibitor of the NF-κB signaling pathway with significant antitumor activity against several human tumor cell lines (GI50 1.38-1.45 µM) and was more potent than paclitaxel against the drug-resistant KBvin cell line. Compound 5a also inhibited LPS-induced NF-κB activation in RAW264.7 cells with an IC50 value of 0.52 µM, prevented IκB-α degradation and p65 nuclear translocation, and suppressed LPS-induced NO production in a dose-dependent manner. The antitumor data in cellular assays indicated that relative positions and types of substituents on the dibenzocyclooctatetraene or acyclic biphenyl as well as torsional angles between the two phenyls are of primary importance to antitumor activity.

[Synthesis and biological evaluation of diarylamines with antitumor activity].

By structural modifications of our previous leads 1-3, 18 diarylamines were designed, synthesized and evaluated with a human tumor cell line panel, including A549, DU145, KB, and KB-vin cell lines, resulting in the discovery of new antitumor agents A6 and B2 with low micromolar G50 values ranging from 1.55-2.10 micromol x L(-1) for above cell lines, and A9 with GI50 values ranging from 1.55-2.10 micromol x L(-1) specially for DU145, KB, and KB-vin cells. Current structure-activity relationships are helpful for further lead optimization.

N-aryl-6-methoxy-1,2,3,4-tetrahydroquinolines: a novel class of antitumor agents targeting the colchicine site on tubulin.

Structural optimizations of the prior lead 1a led to the discovery of a series of N-aryl-6-methoxy-1,2,3,4-tetrahydroquinoline derivatives as a novel class of tubulin polymerization inhibitors targeted at the colchicine binding site. The most active compound 6d showed extremely high cytotoxicity against a human tumor cell line panel (A549, KB, KBvin, and DU145) with GI50 values ranging from 1.5 to 1.7 nM, significantly more potent than paclitaxel, especially against the drug-resistant KBvin cell line, in the same assays. Analogs 5f, 6b, 6c, and 6e were also quite potent, with a GI50 range of 0.011-0.19 µM. In further studies, active compounds 6b-e and 5f significantly inhibited tubulin assembly, with IC50 values of 0.92-1.0 µM and strongly inhibited colchicine binding to tubulin, with inhibition rates of 75-99% (at 5 µM), comparable with or more potent than combretastatin A-4 (IC50 0.96 µM). Current studies included design, synthesis, and biological evaluations of 24 new compounds (series 3-6). Related SAR analysis, molecular modeling, and evaluation of essential drug-like properties, i.e. water solubility, log P, and in vitro metabolic stability, were also performed.

Synthesis and biological evaluation of N-alkyl-N-(4-methoxyphenyl)pyridin-2-amines as a new class of tubulin polymerization inhibitors.

Based on our prior antitumor hits, 32 novel N-alkyl-N-substituted phenylpyridin-2-amine derivatives were designed, synthesized and evaluated for cytotoxic activity against A549, KB, KB(VIN), and DU145 human tumor cell lines (HTCL). Subsequently, three new leads (6a, 7g, and 8c) with submicromolar GI(50) values of 0.19-0.41 µM in the cellular assays were discovered, and these compounds also significantly inhibited tubulin assembly (IC(50) 1.4-1.7 µM) and competitively inhibited colchicine binding to tubulin with effects similar to those of the clinical candidate CA-4 in the same assays. These promising results indicate that these tertiary diarylamine derivatives represent a novel class of tubulin polymerization inhibitors targeting the colchicine binding site and showing significant anti-proliferative activity.