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.

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.

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.

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.

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.

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.

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.

Prodrugs of fluoro-substituted benzoates of EGC as tumor cellular proteasome inhibitors and apoptosis inducers.

The most potent catechin in green tea is (-)-epigallocatechin-3-gallate [(-)-EGCG], which, however, is unstable under physiological conditions. To discover more stable and more potent polyphenol proteasome inhibitors, we synthesized several novel fluoro-substituted (-)-EGCG analogs, named F-EGCG analogs, as well as their prodrug forms with all of -OH groups protected by acetate. We report that the prodrug form of one F-EGCG analog exhibited greater potency than the previously reported peracetate of (-)-EGCG to inhibit proteasomal activity, suppress cell proliferation, and induce apoptosis in human leukemia Jurkat T cells, demonstrating the potential of these compounds to be developed into novel anti-cancer and cancer-preventive agents.

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.

Methylation of green tea polyphenols affects their binding to and inhibitory poses of the proteasome beta5 subunit.

Previously, we showed that ester carbon-containing tea polyphenols, including (-)-epigallocatechin gallate [(-)-EGCG] and (-)-epicatechin-3-gallate [(-)-ECG], potently inhibit proteasomal chymotrypsin-like activity. In addition, our in silico docking study suggested that a particular pose of (-)-EGCG could lead to potential covalent modification of the N-terminal threonine (Thr 1) of the proteasome beta5 subunit in the chymotrypsin-like active site. It has been suggested that some major biotransformation reactions, such as methylation, could result in reduced biological activity of (-)-EGCG in vivo. We hypothesize that methylation reduces binding of (-)-EGCG to the beta5 subunit of the proteasome and, therefore, decreases its proteasomal chymotrypsin-like-inhibitory potency. Here, we report that, while methylation has no effect on nucleophilic susceptibility of (-)-EGCG and (-)-ECG, it may disrupt the ability of these polyphenols to interact with Thr 1 of the proteasome beta5 subunit. In silico docking shows that methylation results in the tea polyphenols' ester carbon being moved away or blocked entirely from Thr 1. Additionally, methylation impairs the ability of (-)-EGCG and (-)-ECG to dock in a consistent low energy pose. These observations, no change in nucleophilic susceptibility, moving or blocking the ester carbon from Thr 1, and lack of a consistent docking pose, suggest that methylation disrupts the ability of (-)-EGCG and (-)-ECG to bind to the proteasome beta5 subunit, which may then diminish their proteasomal chymotrypsin-inhibitory and, therefore, other biological activities.

Evaluation of proteasome-inhibitory and apoptosis-inducing potencies of novel (-)-EGCG analogs and their prodrugs.

The anti-cancer and cancer-preventive effects of green tea and its main constituent (-)-epigallocatechin gallate [(-)-EGCG] are well documented by a variety of studies, including epidemiological, cell culture, animal, and clinical studies. While (-)-EGCG remains the most potent polyphenol in green tea, it is very unstable in neutral or alkaline conditions (i.e. physiologic pH). In an effort to discover more stable polyphenol proteasome inhibitors, we synthesized several novel (-)-EGCG analogs with -OH groups eliminated from the B- and/or D-rings. In addition, we also synthesized their putative prodrugs with -OH groups protected by peracetate that can be removed by cellular cytosolic esterases. We first examined the structure-activity relationship of these unprotected and protected compounds to their proteasome-inhibitory potentials. We found that decreasing -OH groups from either the B- or D-ring leads to diminished proteasome-inhibitory activity in vitro. However, in cultured tumor cells only the protected analogs were capable of potently inhibiting the proteasome activity. Furthermore, these protected analogs induced apoptotic cell death in a tumor cell-specific manner. The superior efficacy of the protected (-)-EGCG analogs indicates the formation of an entirely new compound(s) in intact tumor cells. These data suggest that the B-ring/D-ring peracetate-protected EGCG analogs have great potential to be developed into novel anti-cancer and cancer-preventive agents.

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.

Green tea and tea polyphenols in cancer prevention.

The cancer-preventive effects of green tea and its main constituent (-)-epigallocatechin gallate [(-)-EGCG] are widely supported by results from epidemiological, cell culture, animal and clinical studies in the recent decade. In vitro cell culture studies show that tea polyphenols potently induce apoptotic cell death and cell cycle arrest in tumor cells but not in their normal cell counterparts. Green tea polyphenols affect several signal transduction pathways, including growth factor-mediated, the mitogen-activated protein kinase (MAPK)-dependent, and ubiquitin/proteasome degradation pathways. Epidemiological studies have suggested that the consumption of green tea lowers the risk of cancer. Various animal studies have revealed that treatment by green tea inhibits tumor incidence and multiplicity in different organ sites such as skin, lung, liver, stomach, mammary gland and colon. Phase I and II clinical trials were carried out recently to explore the anticancer effects of green tea in patients with cancer. At this time, more mechanistic research, animal studies, and clinical trials are necessary to further evaluate the role of green tea in cancer prevention.

[Three dimensional quantitative structure-activity relationship of a series of benzocylohepatpyridine farnesyltransferase inhibitors].

AIM: To build a three dimensional structure model that correlates the biological activities and the structures of a series of 1-(8-chloro-6,11-dihydro-5H-benzo[5,6]cyclohepta-[1,2-]pyridin-11-yl) piperazines farnesyl protein transferase (FPTase) inhibitors. METHODS AND RESULTS: Mutation in the ras oncogene takes place in many human cancers, involving 30%-50% of colon and 90% of pancreatic cancer. Ras proteins function as central switches for signals given by growth factors that direct cell growth and cell differentiation. The dependence of the transforming activity of Ras on the farnesylation has led to intense search for FPTase inhibitors that may have therapeutic pontetial as anticancer agents. This paper is to build a three dimensional structural model that correlates the biological activities and the structures of a series of FPTase inhibitors. The investigated sixty-nine inhibitors contain six types of structures, the optimal conformations of which were studied using system search. A three dimensional quantitative structure-activity relationship (3D-QSAR) model was constructed using the method of comparative molecular field analysis (CoMFA). The resulting cross-validation R2 is 0.581, non-cross-validation R2 0.968, SE 0.148, F 198.7. The predicted activities of 10 inhibitors using this 3D-QSAR model are comparable to the experimental activities, indicating that the 3D-QSAR model has ability to predict activities of new inhibitors and offers an approach to design new FPTase inhibitors. CONCLUSION: The information of CoMFA model offers an approach to designing new FPTase inhibitors.

7-imidazolylalkanamido-1-carboxylalkylbenzo-diazepine, a novel series of farnesyltransferase inhibitors.

AIM: Design, synthesis and evaluation of a series of 7-imidazolylalkanamido-1-carboxylalkylbenzodiazepine farnesyltransferase (FTase) inhibitors. METHODS AND RESULTS: Coupling of imidazolylalkylcarboxylic acids and 1-substituted 7-aminobenzodiazepines (5a-5c) yielded 10 new compounds (6-12, 16-18) which were biologically tested against FTase using scintillation proximity assay method. CONCLUSION: Five target compounds were found to be potential farnesyltransferase inhibitors.

Sensitizing human multiple myeloma cells to the proteasome inhibitor bortezomib by novel curcumin analogs.

The proteasome plays a vital role in the degradation of proteins involved in several pathways including the cell cycle, cellular proliferation and apoptosis and is a validated target in cancer treatment. Bortezomib (Velcade®, PS-341) is the first US FDA approved proteasome inhibitor anticancer drug used in the treatment of refractory multiple myeloma. In spite of its improved efficacy compared to alternative therapies, about 60% of patients do not respond to bortezomib due to the emergence of resistance. We hypothesized that novel small molecules could enhance the proteasome-inhibitory and anticancer activities of bortezomib in resistant multiple myeloma cells in vitro and in vivo. The dietary polyphenol curcumin has been shown to exert anti-cancer activity in several cancer cell lines, but the effects of curcumin in solid tumors have been modest primarily due to poor water solubility and poor bioavailability in tissues remote from the gastrointestinal tract. Here we show that the water-soluble analog of curcumin #12, but not curcumin, in combination with bortezomib could enhance the proteasome-inhibitory effect in multiple myeloma cells. Furthermore, the sensitivity of the myeloma cells to cytotoxic killing in the presence of otherwise sublethal concentrations of bortezomib was enhanced by incubation with the curcumin analog #12. These findings justify further investigation into those combinations that may yield potential therapeutic benefit.

[An experimental study of the cervical lymphatic imaging in interstitial magnetic resonance lymphography of tongue using Dextran-DTPA-Gd].

PURPOSE: To explore the application value of the cervical lymphatic imaging in interstitial magnetic resonance lymphography using submucosal injection of Dextran-DTPA-Gd. METHODS: 0.2 mL Dextran-DTPA-Gd (3.96 mmol/L) was injected into the submucosa of the bilateral lingual margins in 12 New Zealand rabbits,and then massaged the injection site for 30 seconds. MR images were obtained before injection and 10, 15, 20, 25, 30, 35, 40, 50, 90 minutes after injection by 3D TOF CE-MRA sequence.The signal intensities of cervical lymph node were measured, the enhancing rates(E%) were calculated and the signal enhancing rates -time curve was drawn. The data was analysed using SPSS11.5 software package. RESULTS: The cervical lymph nodes,the first and second lymphatics were strengthened significantly after injecting Dextran-DTPA-Gd, but the blood vessels were not enhanced at the same time. The enhancing rates of cervical lymph node reached the peak(344%) at 30-min,and the best strengthening effect was achieved between 20-min and 50-min. CONCLUSIONS: As IMRLG contrast agent,the Dextran-DTPA-Gd could image lymphatic drainage lines of the neck and the cervical lymph nodes efficiently.

Synthesis of methylated quercetin derivatives and their reversal activities on P-gp- and BCRP-mediated multidrug resistance tumour cells.

Three methylated quercetins and a series of O-3 substituted 5,7,3',4'-tetra-O-methylated quercetin derivatives have been synthesized and evaluated on the modulating activity of P-gp, BCRP and MRP1 in cancer cell lines. Compound 17 (with a 2-((4-methoxybenzoyl)oxy)ethyl at O-3) is the most potent P-gp modulator. Three derivatives, compound 9 (3,7,3',4'-tetra-O-methylated quercetin), compound 14 (with a 2-((3-oxo-3-(3,4,5trimethoxyphenyl)prop-1-en-1-yl)oxy)ethyl at O-3) and compound 17, consistently exhibited promising BCRP-modulating activity. Interestingly, compound 17 was found to be equipotent against both P-gp and BCRP. Importantly, these synthetic quercetin derivatives did not exhibit any inherent cytotoxicity to cancer cell lines or normal mouse fibroblast cell lines. These quercetin derivatives can be employed as safe and effective modulators of P-gp- or BCRP-mediated drug resistance in cancer.