m6A reader IGF2BP2 promotes lymphatic metastasis by stabilizing DPP4 in papillary thyroid carcinoma.

Lymph node metastasis (LNM) is a major cause of locoregional recurrence of papillary thyroid carcinoma (PTC). However, the mechanisms responsible for LNM are unclear. Aberrant N6-methyladenosine (m6A) RNA modification plays a vital role in cancer progression and metastasis, and whether m6A modification regulates LNM in PTC remains to be determined. This study showed that IGF2BP2 was upregulated in PTC and positively associated with LNM. Functionally, IGF2BP2 knockdown significantly inhibited PTC cell proliferation and invasion in vitro, and vice versa. Moreover, IGF2BP2 knockdown significantly inhibited lymphatic metastasis in vivo. Mechanistically, Human m6A epitranscriptomic microarray, MeRIP, and RIP assays demonstrated that IGF2BP2 activated the NF-KB pathway by enhancing DPP4 stability in an m6A-dependent manner. Furthermore, IGF2BP2 knockdown increased the sensitivity of PTC cells to cisplatin therapy to a certain extent, while its overexpression produced the opposite effects. Overall, this study uncovers that IGF2BP2 promotes lymphatic metastasis via stabilizing DPP4 in an m6A-dependent manner, and provides new insights for understanding the mechanism of lymphatic metastasis in PTC.

Genome-wide association analysis of type II resistance to Fusarium head blight in common wheat.

Background: Fusarium head blight (FHB) is a disease affecting wheat spikes caused by some Fusarium species and leads to cases of severe yield reduction and seed contamination. Identifying resistance genes/QTLs from wheat germplasm may help to improve FHB resistance in wheat production. Methods: Our study evaluated 205 elite winter wheat cultivars for FHB resistance. A high-density 90K SNP array was used for genotyping the panel. A genome-wide association study (GWAS) from cultivars from three different environments was performed using a mixed linear model (MLM). Results: Sixty-six significant marker-trait associations (MTAs) were identified (P < 0.001) on fifteen chromosomes that explained the phenotypic variation ranging from 5.4 to 11.2%. Some important new MTAs in genomic regions involving FHB resistance were found on chromosomes 2A, 3B, 5B, 6A, and 7B. Six MTAs at 92 cM on chromosome 7B were found in cultivars from two different environments. Moreover, there were 11 MTAs consistently associated with diseased spikelet rate and diseased rachis rate as pleiotropic effect loci and D_contig74317_533 on chromosome 5D was novel for FHB resistance. Eight new candidate genes of FHB resistance were predicated in wheat in this study. Three candidate genes, TraesCS5D02G006700, TraesCS6A02G013600, and TraesCS7B02G370700 on chromosome 5DS, 6AS, and 7BL, respectively, were perhaps important in defending against FHB by regulating intramolecular transferase activity, GTP binding, or chitinase activity in wheat, but further validation in needed. In addition, a total of five favorable alleles associated with wheat FHB resistance were discovered. These results provide important genes/loci for enhancing FHB resistance in wheat breeding by marker-assisted selection.

Transcriptome Dynamic Analysis Reveals New Candidate Genes Associated with Resistance to Fusarium Head Blight in Two Chinese Contrasting Wheat Genotypes.

In recent years, Fusarium head blight (FHB) has developed into a global disease that seriously affects the yield and quality of wheat. Effective measures to solve this problem include exploring disease-resistant genes and breeding disease-resistant varieties. In this study, we conducted a comparative transcriptome analysis to identify the important genes that are differentially expressed in FHB medium-resistant (Nankang 1) and FHB medium-susceptible (Shannong 102) wheat varieties for various periods after Fusarium graminearum infection using RNA-seq technology. In total, 96,628 differentially expressed genes (DEGs) were identified, 42,767 from Shannong 102 and 53,861 from Nankang 1 (FDR < 0.05 and |log2FC| > 1). Of these, 5754 and 6841 genes were found to be shared among the three time points in Shannong 102 and Nankang 1, respectively. After inoculation for 48 h, the number of upregulated genes in Nankang 1 was significantly lower than that of Shannong 102, but at 96 h, the number of DEGs in Nankang 1 was higher than that in Shannong 102. This indicated that Shannong 102 and Nankang 1 had different defensive responses to F. graminearum in the early stages of infection. By comparing the DEGs, there were 2282 genes shared at the three time points between the two strains. GO and KEGG analyses of these DEGs showed that the following pathways were associated with disease resistance genes: response to stimulus pathway in GO, glutathione metabolism, phenylpropanoid biosynthesis, plant hormone signal transduction, and plant-pathogen interaction in KEGG. Among them, 16 upregulated genes were identified in the plant-pathogen interaction pathway. There were five upregulated genes, TraesCS5A02G439700, TraesCS5B02G442900, TraesCS5B02G443300, TraesCS5B02G443400, and TraesCS5D02G446900, with significantly higher expression levels in Nankang 1 than in Shannong 102, and these genes may have an important role in regulating the resistance of Nankang 1 to F. graminearum infection. The PR proteins they encode are PR protein 1-9, PR protein 1-6, PR protein 1-7, PR protein 1-7, and PR protein 1-like. In addition, the number of DEGs in Nankang 1 was higher than that in Shannong 102 on almost all chromosomes, except chromosomes 1A and 3D, but especially on chromosomes 6B, 4B, 3B, and 5A. These results indicate that gene expression and the genetic background must be considered for FHB resistance in wheat breeding.

Development and validation of a prognostic nomogram for Hürthle cell thyroid carcinoma: a SEER-based study.

Background: Hürthle cell carcinoma is a rare subtype of thyroid cancer, and its clinical behavior and biological characteristics remain unclear. This study aimed to establish nomogram models for the prognostic evaluation of Hürthle cell thyroid carcinoma (HCTC) in terms of both cancer-specific survival (CSS) and overall survival (OS). Methods: Data for a total of 3,264 patients with HCTC (2004 to 2018) were extracted from the Surveillance, Epidemiology, and End Results (SEER) database. Univariate and multivariate Cox regression analysis was performed to identify significant predictors of prognosis and develop a prognostic nomogram. The performance of the model was assessed based on the area under the receiver operating characteristic curve (AUC), concordance index (c-index), and calibration curves. Results: Multivariate Cox regression analysis showed that age, sex, summary stage, tumor size, N stage, M stage, and treatment with thyroidectomy were independent predictors of OS. Moreover, age, summary stage, tumor size, N stage, M stage, AJCC stage, and treatment with thyroidectomy were significantly correlated with CSS. The c-index of the OS and CSS nomograms developed based on these factors was 0.822 (95% CI: 0.803-0.841) and 0.893 (95% CI: 0.866-0.920), respectively. The AUC was 0.888, 0.841, and 0.834 for 1-, 3-, and 5-year OS and 0.970, 0.949, and 0.933 for 1-, 3-, and 5-year CSS, respectively. The calibration curves showed good agreement between observed and predicted values. Moreover, decision curve analysis revealed that the nomogram had a better clinical utility than individual clinicopathological markers. Conclusions: A prognostic nomogram that allows the individualized assessment of OS and CSS in HCTC was developed. This nomogram could be used to guide treatment decisions in patients with HCTC.

Identification and validation of potential novel biomarkers to predict distant metastasis in differentiated thyroid cancer.

BACKGROUND: Distant metastasis (DM) is not common in differentiated thyroid cancer (DTC). However, it is associated with a significantly poor prognosis. Early detection of high-risk DTC patients is difficult, and the molecular mechanism is still unclear. Therefore, the present study aims to establish a novel predictive model based on clinicopathological parameters and DM-related gene signatures to provide guidelines for clinicians in decision making. METHODS: Weighted gene co-expression network analysis (WGCNA) was performed to discover co-expressed gene modules and hub genes associated with DM. Univariate and multivariate analyses were carried out to identify independent clinicopathological risk factors based on The Cancer Genome Atlas (TCGA) database. An integrated nomogram prediction model was established. Finally, real hub genes were validated using the GSE60542 database and various thyroid cell lines. RESULTS: The midnightblue module was most significantly positively correlated with DM (R=0.56, P=9e-06) by as per WGCNA. DLX5 (AUC: 0.769), COX6B2 (AUC: 0.764), and LYPD1 (AUC: 0.760) were determined to be the real hub genes that play a crucial role in predicting DM. Meanwhile, univariate and multivariate analyses demonstrated that T-stage (OR, 15.03; 95% CI, 1.75-319.40; and P=0.024), histologic subtype (OR, 0.17; 95% CI, 0.03-0.92; and P=0.042) were the independent predictors of DM. Subsequently, a nomogram model was constructed based on gene signatures and independent clinical risk factors exhibited good performance. Additionally, the mRNA expressions of real hub genes in the GSE60542 dataset were consistent with TCGA. CONCLUSIONS: The present study has provided a reliable model to predict DM in patients with DTC. This model is likely to serve as an individual risk assessment tool in therapeutic decision-making.

Genome wide association study of the whiteness and colour related traits of flour and dough sheets in common wheat.

Flour whiteness and colour are important factors that influence the quality of wheat flour and end-use products. In this study, a genome wide association study focusing on flour and dough sheet colour using a high density genetic map constructed with 90K single nucleotide polymorphism arrays in a panel of 205 elite winter wheat accessions was conducted in two different locations in 2 years. Eighty-six significant marker-trait associations (MTAs) were detected for flour whiteness and the brightness index (L* value), the redness index (a* value), and the yellowness index (b* value) of flour and dough sheets (P < 10-4) on homologous group 1, 2, 5 and 7, and chromosomes 3A, 3B, 4A, 6A and 6B. Four, three, eleven, eleven MTAs for the flour whiteness, L* value, a* value, b* value, and one MTA for the dough sheet L* value were identified in more than one environment. Based on MATs, some important new candidate genes were identified. Of these, two candidate genes, TraesCS5D01G004300 and Gsp-1D, for BS00000020_51 were found in wheat, relating to grain hardness. Other candidate genes were associated with proteins, the fatty acid biosynthetic process, the ketone body biosynthetic process, etc.

The Role of m6A RNA Methylation-Related lncRNAs in the Prognosis and Tumor Immune Microenvironment of Papillary Thyroid Carcinoma.

Emerging evidence has indicated that N6-methylandenosine (m6A) RNA methylation plays a critical role in cancer development. However, the function of m6A RNA methylation-related long noncoding RNAs (m6A-lncRNAs) in papillary thyroid carcinoma (PTC) has never been reported. This study aimed to investigate the role of m6A-lncRNAs in the prognosis and tumor microenvironment (TME) of PTC. Three subgroups (clusters 1, 2, and 3) were identified by consensus clustering of 19 prognosis-related m6A-lncRNA regulators, of which cluster 1 is preferentially related to unfavorable prognosis, lower immune scores, and distinct immune infiltrate level. A risk-score model was established based on 8 prognosis-related m6A-lncRNAs. Patients with a high-risk score showed a worse prognosis, and the ROC indicated a reliable prediction performance for patients with PTC (AUC = 0.802). As expected, the immune scores, the infiltration levels of immune cells, and ESTIMATE scores in the low-risk subgroups were notably higher (p < 0.001) when compared with those in high-risk subgroups. Furthermore, GSEA analysis revealed that tumor associated pathways, hallmarks, and biological processes were remarkably enriched in the high-risk subgroup. Further analysis indicated that the risk score and age were independent prognostic factors for PTC. An integrated nomogram was constructed that accurately predicted the survival status (AUC = 0.963). Moreover, a lncRNA-miRNA-mRNA regulated network was established based on seven prognosis-related m6A-lncRNAs. In addition, 30 clinical samples and different PTC cells were validated. This is the first study to reveal that m6A-lncRNAs plays a vital role in the prognosis and TME of PTC. To a certain degree, m6A-lncRNAs can be considered as new, promising prognostic biomarkers and treatment targets.

Management of Lateral Multiple-Level Metastasis in N1b Papillary Thyroid Microcarcinoma.

Background: The optimal extent of therapeutic lateral neck dissection (LND) in the management of N1b papillary thyroid microcarcinoma (PTMC) is still under debate in clinical practice. In this light, our study aims to explore the incidence, patterns, and predictive factors of the lateral multiple-level metastasis in N1b PTMC patients. Methods: The clinical records of 142 patients diagnosed with N1b PTMC who underwent therapeutic LND from July 2015 to November 2018 at our institution were retrospectively reviewed. Univariate and multivariate analyses were conducted to examine the predictive factors associated with lateral multiple-level metastasis. The recurrence-free survival was analyzed and confirmed by Kaplan-Meier plots and log-rank test. Results: The overall frequency of lateral multiple-level metastasis was 50.7% in N1b PTMC patients, and two-level to four-level simultaneous metastasis were present in 26.8, 17.6, and 6.3% patients, respectively. Extrathyroidal extension (ETE) (OR = 5.79, 95% CI, 1.36-24.59; P = 0.017) and the central metastatic lymph node ratio (CLNR) with values equal or higher than 0.61 (OR = 6.18, 95% CI, 2.53-15.09; P < 0.001) served as independent predictors of multiple-level metastasis in N1b PTMC patients. Moreover, locoregional recurrence was significantly higher in the selective neck dissection (SND) group compared to the modified radical neck dissection (MRND) one (HR = 3.65, 95% CI, 1.11-12.00; P = 0.03). Conclusion: Our results show that the lateral multiple-level metastasis was relatively common, and we suggest MRND to be considered for N1b PTMC patients with ETE or CLNR equal or higher than 0.61.

A New Class of Safe, Potent, and Specific P-gp Modulator: Flavonoid Dimer FD18 Reverses P-gp-Mediated Multidrug Resistance in Human Breast Xenograft in Vivo.

Flavonoid dimer FD18 is a new class of dimeric P-gp modulator that can reverse cancer drug resistance. FD18 is a potent (EC50 = 148 nM for paclitaxel), safe (selective index = 574), and selective P-glycoprotein (P-gp) modulator. FD18 can modulate multidrug resistance toward paclitaxel, vinblastine, vincristine, doxorubicin, daunorubicin, and mitoxantrone in human breast cancer LCC6MDR in vitro. FD18 (1 µM) can revert chemosensitivity of LCC6MDR back to parental LCC6 level. FD18 was 11- to 46-fold more potent than verapamil. FD18 (1 µM) can increase accumulation of doxorubicin by 2.7-fold, daunorubicin (2.1-fold), and rhodamine 123 (5.2-fold) in LCC6MDR. FD18 inhibited P-gp-mediated doxorubicin efflux and has no effect on influx. FD18 at 1 µM did not affect the protein expression level of P-gp. Pharmacokinetics studies indicated that intraperitoneal administration of 45 mg/kg FD18 was enough to maintain a plasma level above EC50 (148 nM) for more than 600 min. Toxicity studies with FD18 (90 mg/kg, i.p. for 12 times in 22 days) with paclitaxel (12 mg/kg, i.v. for 12 times in 22 days) revealed no obvious toxicity or death in mice. In vivo efficacy studies indicated that FD18 (45 mg/kg, i.p. for 12 times in 22 days) together with paclitaxel (12 mg/kg, i.v. for 12 times in 22 days) resulted in a 46% reduction in LCC6MDR xenograft volume (n = 11; 648 ± 84 mm(3)) compared to paclitaxel control (n = 8; 1201 ± 118 mm(3)). There were no animal deaths or significant drop in body weight and vital organ wet weight. FD18 can increase paclitaxel accumulation in LCC6MDR xenograft by 1.8- to 2.2-fold. The present study suggests that FD18 represents a new class of safe and potent P-gp modulator in vivo.

Radiation dose reduction in medical x-ray CT via Fourier-based iterative reconstruction.

PURPOSE: A Fourier-based iterative reconstruction technique, termed Equally Sloped Tomography (EST), is developed in conjunction with advanced mathematical regularization to investigate radiation dose reduction in x-ray CT. The method is experimentally implemented on fan-beam CT and evaluated as a function of imaging dose on a series of image quality phantoms and anonymous pediatric patient data sets. Numerical simulation experiments are also performed to explore the extension of EST to helical cone-beam geometry. METHODS: EST is a Fourier based iterative algorithm, which iterates back and forth between real and Fourier space utilizing the algebraically exact pseudopolar fast Fourier transform (PPFFT). In each iteration, physical constraints and mathematical regularization are applied in real space, while the measured data are enforced in Fourier space. The algorithm is automatically terminated when a proposed termination criterion is met. Experimentally, fan-beam projections were acquired by the Siemens z-flying focal spot technology, and subsequently interleaved and rebinned to a pseudopolar grid. Image quality phantoms were scanned at systematically varied mAs settings, reconstructed by EST and conventional reconstruction methods such as filtered back projection (FBP), and quantified using metrics including resolution, signal-to-noise ratios (SNRs), and contrast-to-noise ratios (CNRs). Pediatric data sets were reconstructed at their original acquisition settings and additionally simulated to lower dose settings for comparison and evaluation of the potential for radiation dose reduction. Numerical experiments were conducted to quantify EST and other iterative methods in terms of image quality and computation time. The extension of EST to helical cone-beam CT was implemented by using the advanced single-slice rebinning (ASSR) method. RESULTS: Based on the phantom and pediatric patient fan-beam CT data, it is demonstrated that EST reconstructions with the lowest scanner flux setting of 39 mAs produce comparable image quality, resolution, and contrast relative to FBP with the 140 mAs flux setting. Compared to the algebraic reconstruction technique and the expectation maximization statistical reconstruction algorithm, a significant reduction in computation time is achieved with EST. Finally, numerical experiments on helical cone-beam CT data suggest that the combination of EST and ASSR produces reconstructions with higher image quality and lower noise than the Feldkamp Davis and Kress (FDK) method and the conventional ASSR approach. CONCLUSIONS: A Fourier-based iterative method has been applied to the reconstruction of fan-bean CT data with reduced x-ray fluence. This method incorporates advantageous features in both real and Fourier space iterative schemes: using a fast and algebraically exact method to calculate forward projection, enforcing the measured data in Fourier space, and applying physical constraints and flexible regularization in real space. Our results suggest that EST can be utilized for radiation dose reduction in x-ray CT via the readily implementable technique of lowering mAs settings. Numerical experiments further indicate that EST requires less computation time than several other iterative algorithms and can, in principle, be extended to helical cone-beam geometry in combination with the ASSR method.

High-resolution, low-dose phase contrast X-ray tomography for 3D diagnosis of human breast cancers.

Mammography is the primary imaging tool for screening and diagnosis of human breast cancers, but ~10-20% of palpable tumors are not detectable on mammograms and only about 40% of biopsied lesions are malignant. Here we report a high-resolution, low-dose phase contrast X-ray tomographic method for 3D diagnosis of human breast cancers. By combining phase contrast X-ray imaging with an image reconstruction method known as equally sloped tomography, we imaged a human breast in three dimensions and identified a malignant cancer with a pixel size of 92 µm and a radiation dose less than that of dual-view mammography. According to a blind evaluation by five independent radiologists, our method can reduce the radiation dose and acquisition time by ~74% relative to conventional phase contrast X-ray tomography, while maintaining high image resolution and image contrast. These results demonstrate that high-resolution 3D diagnostic imaging of human breast cancers can, in principle, be performed at clinical compatible doses.

Propulsion of African trypanosomes is driven by bihelical waves with alternating chirality separated by kinks.

Trypanosoma brucei, a parasitic protist with a single flagellum, is the causative agent of African sleeping sickness. Propulsion of T. brucei was long believed to be by a drill-like, helical motion. Using millisecond differential interference-contrast microscopy and analyzing image sequences of cultured procyclic-form and bloodstream-form parasites, as well as bloodstream-form cells in infected mouse blood, we find that, instead, motility of T. brucei is by the propagation of kinks, separating left-handed and right-handed helical waves. Kink-driven motility, previously encountered in prokaryotes, permits T. brucei a helical propagation mechanism while avoiding the large viscous drag associated with a net rotation of the broad end of its tapering body. Our study demonstrates that millisecond differential interference-contrast microscopy can be a useful tool for uncovering important short-time features of microorganism locomotion.

Modulation of multidrug resistance protein 1 (MRP1/ABCC1)-mediated multidrug resistance by bivalent apigenin homodimers and their derivatives.

Here we showed that bivalency approach is effective in modulating multidrug resistance protein 1 (MRP1/ABCC1)-mediated doxorubicin (DOX) and etoposide (VP16) resistance in human 2008/MRP1 ovarian carcinoma cells. Flavonoid dimers bearing five or six ethylene glycol (EG) units with 6-methyl (4e, 4f) or 7-methyl (5e, 5f) substitution on the ring A of flavonoid dimers have the highest modulating activity for DOX against MRP1 with an EC(50) ranging from 73 to 133 nM. At 0.5 microM, the flavonoid dimer 4e was sufficient to restore DOX accumulation in 2008/MRP1 to parental 2008/P level. Lineweaver-Burk and Dixon plot suggested that it is likely a competitive inhibitor of DOX transport with a K(i) = 0.2 microM. Our data suggest that flavonoid dimers have a high affinity toward binding to DOX recognition site of MRP1. This results in inhibiting DOX transport, increasing intracellular DOX retention, and finally resensitizing 2008/MRP1 to DOX. The present study demonstrates that flavonoid dimers can be employed as an effective modulator of MRP1-mediated drug resistance in cancer cells.

Quinacrine and a novel apigenin dimer can synergistically increase the pentamidine susceptibility of the protozoan parasite Leishmania.

OBJECTIVES: The aim of this study was to investigate the synergistic effect of quinacrine and a novel apigenin dimer (compound 9d) on reversing pentamidine resistance of Leishmania parasites. METHODS: Pentamidine-resistant cell lines, LePentR50 and LdAG83PentR50, were generated by gradually increasing pentamidine pressure on wild-type promastigotes. We tested the effects of different combinations of quinacrine and an apigenin dimer on modulating the pentamidine resistance levels of LePentR50 and LdAG83PentR50 using an MTS proliferation assay. We then measured the accumulation level of pentamidine using HPLC. The fractional inhibitory concentration index (FICI) method was used to evaluate the interaction between quinacrine and the apigenin dimer on reversing pentamidine resistance in Leishmania. RESULTS: LePentR50 and LdAG83PentR50 promastigotes were approximately 8.6- and 4.6-fold more resistant to pentamidine than their wild-type parents. Amastigotes derived from LePentR50 and LdAG83PentR50 were also pentamidine-resistant. We found that quinacrine can increase the susceptibility of Leishmania to pentamidine. Quinacrine, when used at 6 microM, can increase the IC(50) of pentamidine by 3.8-, 3.4-, 3.5- and 6.3-fold in wild-type Leishmania enriettii Le, LePentR50, wild-type Leishmania donovani LdAG83 and LdAG83PentR50, respectively. Quinine, quinidine and verapamil did not show any sensitizing effect. The sensitizing effect of quinacrine was: (i) dose-dependent; (ii) not associated with an increase in pentamidine accumulation; and (iii) only observed in pentamidine-resistant but not sodium stibogluconate-resistant or vinblastine-resistant parasites. Other than quinacrine, we also found that an apigenin dimer (compound 9d), previously shown to be able to inhibit ABCB1-mediated cancer drug resistance in mammalian cells, can also increase the pentamidine susceptibility of Leishmania. 9d, when used at 6 microM, can increase the IC(50) of pentamidine by 2.5-, 4.2-, 1.6- and 1.9-fold in Le, LePentR50, LdAG83 and LdAG83PentR50, respectively. Unlike quinacrine, sensitization by 9d was accompanied by an increase in pentamidine accumulation, presumably due to the inhibition of an ABC transporter. Using the FICI method, we found that quinacrine and 9d can act synergistically. When they are used in a 1:1 ratio, they sensitize LePentR50 to pentamidine by 19-fold, with an FICI of 0.48 (P < 0.005), indicating that they might act synergistically. CONCLUSIONS: Our findings support the notion that the pentamidine susceptibility of Leishmania is mediated by multiple targets. Quinacrine and apigenin dimer 9d, each inhibiting its own target, can have a synergistic effect when used together to sensitize Leishmania to pentamidine.

Flavonoid dimers as bivalent modulators for p-glycoprotein-based multidrug resistance: structure-activity relationships.

We recently described the modulatory activities of apigenin homodimers linked by ethylene glycol units in multidrug- resistant breast cancer and leukemic cells overexpressing ABCB1 (P-glycoprotein, P-gp). To further improve the potency of these dimers, a small library of flavonoid homodimers and heterodimers were synthesized, and their in vitro activity in reversing cellular resistance to paclitaxel, along with structure-activity relationships (SAR), were evaluated using a P-gp-expressing human breast cancer cell line. Among these synthesized homodimers, many showed more potent reversing activity than that of the parent compound and verapamil. Two compounds in particular showed promising reversing activity at sub-micromolar concentrations with no cytotoxic effects. Regarding SAR trends, flavonoid dimers with nonpolar and hydrophobic substituents (e.g., methyl and ethyl groups) generally showed more potent resistance-reversing activity than that of dimers with polar and hydrophilic substituents (e.g. hydroxy groups) at the C3, C6, and C7 positions, but not at C5. In terms of substituent steric bulk at C6, it was found that the flavonoid dimer with methyl groups was optimal, with bulkier substituents leading to lower reversing activity. Comparisons of flavonoid heterodimers with the corresponding homodimers revealed that the two binding sites on P-gp for flavonoid moieties are quite similar to each other. Besides paclitaxel, these new compounds also increased drug accumulation and enhanced the cytotoxicity of other cancer drugs such as doxorubicin, vincristine, and vinblastine by decreasing the IC(50) values 4-45-fold.

Flavonoid dimers as bivalent modulators for pentamidine and sodium stiboglucanate resistance in leishmania.

Drug resistance by overexpression of ATP-binding cassette (ABC) transporters is an impediment in the treatment of leishmaniasis. Flavonoids are known to reverse multidrug resistance (MDR) in Leishmania and mammalian cancers by inhibiting ABC transporters. Here, we found that synthetic flavonoid dimers with three (compound 9c) or four (compound 9d) ethylene glycol units exhibited a significantly higher reversing activity than other shorter or longer ethylene glycol-ligated dimers, with approximately 3-fold sensitization of pentamidine and sodium stibogluconate (SSG) resistance in Leishmania, respectively. This modulatory effect was dosage dependent and not observed in apigenin monomers with the linker, suggesting that the modulatory effect is due to its bivalent nature. The mechanism of reversal activity was due to increased intracellular accumulation of pentamidine and total antimony in Leishmania. Compared to other MDR modulators such as verapamil, reserpine, quinine, quinacrine, and quinidine, compounds 9c and 9d were the only agents that can reverse SSG resistance. In terms of reversing pentamidine resistance, 9c and 9d have activities comparable to those of reserpine and quinacrine. Modulators 9c and 9d exhibited reversal activity on pentamidine resistance among LeMDR1(-/-), LeMDR1(+/+), and LeMDR1-overexpressed mutants, suggesting that these modulators are specific to a non-LeMDR1 pentamidine transporter. The LeMDR1 copy number is inversely related to pentamidine resistance, suggesting that it might be involved in importing pentamidine into the mitochondria. In summary, bivalency could be a useful strategy for the development of more potent ABC transporter modulators and flavonoid dimers represent a promising reversal agent for overcoming pentamidine and SSG resistance in parasite Leishmania.

Flavonoid dimers as bivalent modulators for P-glycoprotein-based multidrug resistance: synthetic apigenin homodimers linked with defined-length poly(ethylene glycol) spacers increase drug retention and enhance chemosensitivity in resistant cancer cells.

Much effort has been spent on searching for better P-glycoprotein- (P-gp-) based multidrug resistance (MDR) modulators. Our approach was to target the binding sites of P-gp using dimers of dietary flavonoids. A series of apigenin-based flavonoid dimers, linked by poly(ethylene glycol) chains of various lengths, have been synthesized. These flavonoid dimers modulate drug chemosensitivity and retention in breast and leukemic MDR cells with the optimal number of ethylene glycol units equal to 2-4. Compound 9d bearing four ethylene glycol units increased drug accumulation in drug-resistant cells and enhanced cytotoxicity of paclitaxel, doxorubicin, daunomycin, vincristine, and vinblastine in drug-resistant breast cancer and leukemia cells in vitro, resulting in reduction of IC50 by 5-50 times. This compound also stimulated P-gp's ATPase activity by 3.3-fold. Its modulating activity was presumably by binding to the substrate binding sites of P-gp and disrupting drug efflux.

Pseudolaric acid B, a novel microtubule-destabilizing agent that circumvents multidrug resistance phenotype and exhibits antitumor activity in vivo.

PURPOSE: Pseudolaric acid B (PAB) is the major bioactive constituent in the root bark of Pseudolarix kaempferi that has been used as an antifungal remedy in traditional Chinese medicine. Previous studies showed that PAB exhibited substantial cytotoxicity. The aims of this study were to elucidate the molecular target of PAB, to examine its mechanism of action, and to evaluate the efficacy of this compound in vivo. EXPERIMENTAL DESIGN: The effect of PAB on cell growth inhibition toward a panel of cancer cell lines was assayed. Cell cycle analysis, Western blotting, immunocytochemistry, and apoptosis analysis were carried out to examine the mechanism of action. Tubulin polymerization assays were conducted to examine the interaction between PAB and tubulin. A P-glycoprotein-overexpressing cell line was used to evaluate the efficacy of PAB toward multidrug-resistant phenotypes. In vivo efficacy of PAB was evaluated by the murine xenograft model. RESULTS: PAB induces cell cycle arrest at G2-M transition, leading to apoptosis. The drug disrupts cellular microtubule networks and inhibits the formation of mitotic spindles. Polymerization of purified bovine brain tubulin was dose-dependently inhibited by PAB. Furthermore, PAB circumvents the multidrug resistance mechanism, displaying notable potency also in P-glycoprotein-overexpressing cells. Finally, we showed that PAB is effective in inhibiting tumor growth in vivo. CONCLUSIONS: We identified the microtubules as the molecular target of PAB. Furthermore, we showed that PAB circumvents P-glycoprotein overexpression-induced drug resistance and is effective in inhibiting tumor growth in vivo. Our work will facilitate the future development of PAB as a cancer therapeutic.