Manipulating Diastereomeric Bicyclononynes to Sensitively Determine Enzyme Activity and Facilitate Macromolecule Conjugations.

Bicyclo[6.1.0]nonyne (BCN) is one of the most commonly used cycloalkynes in strain-promoted azide-alkyne cycloaddition (SPAAC). The synthesis of BCN produces two diastereomers, exo-BCN and endo-BCN. The potential significance of the different steric structures of the tricyclic fused rings in SPAAC products synthesized from the BCN diastereomers has not been previously studied. We first demonstrated that only endo-BCN could reduce the level of fluorescence quenching in SPAAC reaction products. The reduction was likely due to the presence of extended tricyclic fused ring systems. This hypothesis was supported by the synthesis of a fluorescence always-on construct by substituting endo-BCN for exo-BCN in a previously reported chemical probe that was characterized with good contact fluorescence quenching. We also synthesized bis-BCN derivatives to enhance the steric structural differences in the corresponding SPAAC products. A constitutional isomer of the azido-derivatized 5(6)-carboxyfluorescein [5(6)-FAM] was reacted with both bis-exo-BCN and bis-endo-BCN compounds. However, one form of the bis-exo-BCN-based product did not augment contact fluorescence quenching, while a second bis-exo-BCN product could not further reduce contact fluorescence quenching. Nevertheless, a new fluorescence turn-on chemical probe was employed to determine the activities of two serum biomarkers, butyrylcholinesterase and paraoxonase 1. Moreover, bis-endo-BCN was exploited to successfully conjugate BSA with a 5-FAM derivative compound.

Characterization of the pH-dependent protein stability of 3α-hydroxysteroid dehydrogenase/carbonyl reductase by differential scanning fluorimetry.

The characterization of protein stability is essential for understanding the functions of proteins. Hydroxysteroid dehydrogenase is involved in the biosynthesis of steroid hormones and the detoxification of xenobiotic carbonyl compounds. However, the stability of hydroxysteroid dehydrogenases has not yet been characterized in detail. Here, we determined the changes in Gibbs free energy, enthalpy, entropy, and heat capacity of unfolding for 3α-hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) by varying the pH and urea concentration through differential scanning fluorimetry and presented pH-dependent protein stability as a function of temperature. 3α-HSD/CR shows the maximum stability of 30.79 kJ mol-1 at 26.4°C, pH 7.6 and decreases to 7.74 kJ mol-1 at 25.7°C, pH 4.5. The change of heat capacity of 30.25 ± 1.38 kJ mol-1 K-1 is obtained from the enthalpy of denaturation as a function of melting temperature at varied pH. Two proton uptakes are linked to protein unfolding from residues with differential pKa of 4.0 and 6.5 in the native and denatured states, respectively. The large positive heat capacity change indicated that hydrophobic interactions played an important role in the folding of 3α-HSD/CR. These studies reveal the mechanism of protein unfolding in HSD and provide a convenient method to extract thermodynamic parameters for characterizing protein stability using differential scanning fluorimetry.

Time-dependent effects of storage at -80 °C on the stability of butyrylcholinesterase activity in human serum.

Objectives: Butyrylcholinesterase (BChE) is an important biomarker in serum, and aberrant BChE activity indicates onset and progression of human diseases. The duration of serum storage at -80 °C may introduce variability into and compromise the reproducibility of BChE activity measurements. Design and Methods: We collected serum samples from eight healthy volunteers and determined serum BChE activity in these samples using a sensitive fluorescence assay at various time points during a six-month storage period at -80 °C. Changes in averaged BChE activity over storage time were assessed by repeated measures analysis of variance (ANOVA). Sidak multiple comparisons test was also used to perform post-hoc analysis. Results: Almost all determined BChE activity values lay within the normal physiological range of BChE activity. However, repeated measures ANOVA using mean BChE activity vs. storage time showed that BChE activity values from two time points were significantly different. Analysis by Sidak multiple comparisons test provided no substantial change of BChE activity during the first 90 days of storage, but BChE activity noticeably decreased after 90 days. Conclusions: Serum samples stored in -80 °C for up to 90 days can be exploited to accurately determine BChE activity.

Sensitive Assay for the Lactonase Activity of Serum Paraoxonase 1 (PON1) by Harnessing the Fluorescence Turn-On Characteristics of Bioorthogonally Synthesized and Geometrically Controlled Chemical Probes.

The lactonase activity of paraoxonase 1 (PON1) has a crucial antiatherogenic function, and also serves as an important biochemical marker in human blood because the aberrant lactonase activity of PON1 is a key indicator for a number of diverse human diseases. However, no sensitive fluorescence assays that detect PON1 lactonase activity are available. We report the synthesis of two fluorescence turn-on chemical probes 16a and 16b (16) able to quantify PON1 lactonase activity. The chemical probes were constructed utilizing a disulfide-containing bicyclononyne, derivatives of rhodamine B and carboxyfluorescein, and reactions including copper-free azide-alkyne cycloaddition. Fluorescence quenching in 16 was characterized by spectroscopic studies and was mainly attributed to the effect of contact quenching. Kinetic analysis of 16b confirmed the outstanding reactivity and specificity of 16b with thiols in the presence of general base catalysts. The 16b-based assay was employed to determine PON1 lactonase activity, with a linear range of 10.8-232.1 U L-1 and detection limit (LOD) of 10.8 U L-1, to quantify serum PON1 activity in human sera, and to determine the Ki of 20.9 µM for the 2-hydroxyquinoline inhibition of PON1 lactonase. We are employing 16b to develop high-throughput assays for PON1 lactonase activity.

The Double-Edged Sword of Beta2-Microglobulin in Antibacterial Properties and Amyloid Fibril-Mediated Cytotoxicity.

Beta2-microglobulin (B2M) a key component of major histocompatibility complex class I molecules, which aid cytotoxic T-lymphocyte (CTL) immune response. However, the majority of studies of B2M have focused only on amyloid fibrils in pathogenesis to the neglect of its role of antimicrobial activity. Indeed, B2M also plays an important role in innate defense and does not only function as an adjuvant for CTL response. A previous study discovered that human aggregated B2M binds the surface protein structure in Streptococci, and a similar study revealed that sB2M-9, derived from native B2M, functions as an antibacterial chemokine that binds Staphylococcus aureus. An investigation of sB2M-9 exhibiting an early lymphocyte recruitment in the human respiratory epithelium with bacterial challenge may uncover previously unrecognized aspects of B2M in the body's innate defense against Mycobactrium tuberculosis. B2M possesses antimicrobial activity that operates primarily under pH-dependent acidic conditions at which B2M and fragmented B2M may become a nucleus seed that triggers self-aggregation into distinct states, such as oligomers and amyloid fibrils. Modified B2M can act as an antimicrobial peptide (AMP) against a wide range of microbes. Specifically, these AMPs disrupt microbe membranes, a feature similar to that of amyloid fibril mediated cytotoxicity toward eukaryotes. This study investigated two similar but nonidentical effects of B2M: the physiological role of B2M, in which it potentially acts against microbes in innate defense and the role of B2M in amyloid fibrils, in which it disrupts the membrane of pathological cells. Moreover, we explored the pH-governing antibacterial activity of B2M and acidic pH mediated B2M amyloid fibrils underlying such cytotoxicity.

Functional role of residues involved in substrate binding of human 4-hydroxyphenylpyruvate dioxygenase.

4-Hydroxylphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4-hydroxylphenylpyruvate (HPP) to homogentisate, the important step for tyrosine catabolism. Comparison of the structure of human HPPD with the substrate-bound structure of A. thaliana HPPD revealed notably different orientations of the C-terminal helix. This helix performed as a closed conformation in human enzyme. Simulation revealed a different substrate-binding mode in which the carboxyl group of HPP interacted by a H-bond network formed by Gln334, Glu349 (the metal-binding ligand), and Asn363 (in the C-terminal helix). The 4-hydroxyl group of HPP interacted with Gln251 and Gln265. The relative activity and substrate-binding affinity were preserved for the Q334A mutant, implying the alternative role of Asn363 for HPP binding and catalysis. The reduction in kcat/Km of the Asn363 mutants confirmed the critical role in catalysis. Compared to the N363A mutant, the dramatic reduction in the Kd and thermal stability of the N363D mutant implies the side-chain effect in the hinge region rotation of the C-terminal helix. The activity and binding affinity were not recovered by double mutation; however, the 4-hydroxyphenylacetate intermediate formation by the uncoupled reaction of Q334N/N363Q and Q334A/N363D mutants indicated the importance of the H-bond network in the electrophilic reaction. These results highlight the functional role of the H-bond network in a closed conformation of the C-terminal helix to stabilize the bound substrate. The extremely low activity and reduction in Q251E's Kd suggest that interaction coupled with the H-bond network is crucial to locate the substrate for nucleophilic reaction.

Thermodynamic analysis of remote substrate binding energy in 3α-hydroxysteroid dehydrogenase/carbonyl reductase catalysis.

The binding energy of enzyme and substrate is used to lower the activation energy for the catalytic reaction. 3α-HSD/CR uses remote binding interactions to accelerate the reaction of androsterone with NAD+. Here, we examine the enthalpic and entropic components of the remote binding energy in the 3α-HSD/CR-catalyzed reaction of NAD+ with androsterone versus the substrate analogs, 2-decalol and cyclohexanol, by analyzing the temperature-dependent kinetic parameters through steady-state kinetics. The effects of temperature on kcat/Km for 3α-HSD/CR acting on androsterone, 2-decalol, and cyclohexanol show the reactions are entropically favorable but enthalpically unfavorable. Thermodynamic analysis from the temperature-dependent values of Km and kcat shows the binding of the E-NAD+ complex with either 2-decalol or cyclohexanol to form the ternary complex is endothermic and entropy-driven, and the subsequent conversion to the transition state is both enthalpically and entropically unfavorable. Hence, solvation entropy may play an important role in the binding process through both the desolvation of the solute molecules and the release of bound water molecules from the active site into bulk solvent. As compared to the thermodynamic parameters of 3α-HSD/CR acting on cyclohexanol, the hydrophobic interaction of the B-ring of steroids with the active site of 3α-HSD/CR contributes to catalysis by increasing exclusively the entropy of activation (ΔTΔS‡â€¯= 1.8 kcal/mol), while the BCD-ring of androsterone significantly lowers ΔΔH‡ by 10.4 kcal/mol with a slight entropic penalty of -1.9 kcal/mol. Therefore, the remote non-reacting sites of androsterone may induce a conformational change of the substrate binding loop with an entropic cost for better interaction with the transition state to decrease the enthalpy of activation, significantly increasing catalytic efficiency.

Corroboration of Zn(ii)-Mg(ii)-tertiary structure interplays essential for the optimal catalysis of a phosphorothiolate thiolesterase ribozyme.

The TW17 ribozyme, a catalytic RNA selected from a pool of artificial RNA, is specific for the Zn2+-dependent hydrolysis of a phosphorothiolate thiolester bond. Here, we describe the organic synthesis of both guanosine α-thio-monophosphate and the substrates required for selecting and characterizing the TW17 ribozyme, and for deciphering the catalytic mechanism of the ribozyme. By successively substituting the substrate originally conjugated to the RNA pool with structurally modified substrates, we demonstrated that the TW17 ribozyme specifically catalyzes phosphorothiolate thiolester hydrolysis. Metal titration studies of TW17 ribozyme catalysis in the presence of Zn2+ alone, Zn2+ and Mg2+, and Zn2+ and [Co(NH3)6]3+ supported our findings that Zn2+ is absolutely required for ribozyme catalysis, and indicated that optimal ribozyme catalysis involves the presence of outer-sphere and one inner-sphere Mg2+. A survey of the TW17 ribozyme activity at various pHs revealed that the activity of the ribozyme critically depends on the alkaline conditions. Moreover, a GNRA tetraloop-containing ribozyme constructed with active catalysis in trans provided catalysis and multiple substrate turnover efficiencies significantly higher than ribozymes lacking a GNRA tetraloop. This research supports the essential roles of Zn2+, Mg2+, and a GNRA tetraloop in modulating the TW17 ribozyme structure for optimal ribozyme catalysis, leading also to the formulation of a proposed reaction mechanism for TW17 ribozyme catalysis.

Contribution of remote substrate binding energy to the enzymatic rate acceleration for 3α-hydroxysteroid dehydrogenase/carbonyl reductase.

3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) catalyzes the oxidation of androsterone with NAD+ to form androstanedione and NADH with the rate limiting step being the release of NADH. In this study, we elucidate the role of remote substrate binding interactions contributing to the rate enhancement by 3α-HSD/CR through steady-state kinetic studies with the truncated substrate analogs. No enzyme activity was detected for methanol, ethanol, and 2-propanol, which lack the steroid scaffold of androsterone, implying that the steroid scaffold plays an important role in enzyme catalytic specificity. As compared to cyclohexanol, the activity for 2-decalol, androstenol, and androsterone increases by 0.9-, 90-, and 200-fold in kcat, and 37-, 1.9 × 106-, and 1.8 × 106-fold in kcat/KB, respectively. The rate limiting step is hydride transfer for 3α-HSD/CR catalyzing the reaction of cyclohexanol with NAD+ based on the observed rapid equilibrium ordered mechanism and equal deuterium isotope effects of 3.9 on V and V/K for cyclohexanol. The kcat/KB value results in ΔG‡ of 14.7, 12.6, 6.2, and 6.2 kcal/mol for the 3α-HSD/CR catalyzed reaction of cyclohexanol, 2-decalol, androstenol, and androsterone, respectively. Thus, the uniform binding energy from the B-ring of steroids with the active site of 3α-HSD/CR equally contributes 2.1 kcal/mol to stabilize both the transition state and ground state of the ternary complex, leading to the similarity in kcat for 2-decalol and cyclohexanol. Differential binding interactions of the remote BCD-ring and CD-ring of androsterone with the active site of 3α-HSD/CR contribute 8.5 and 6.4 kcal/mol to the stabilization of the transition state, respectively. The removal of the carbonyl group at C17 of androsterone has small effects on catalysis. Both uniform and differential binding energies from the remote sites of androsterone compared to cyclohexanol contribute to the 3α-HSD/CR catalysis, resulting in the increases in kcat and kcat/KB.

Azide-alkyne cycloaddition for universal post-synthetic modifications of nucleic acids and effective synthesis of bioactive nucleic acid conjugates.

The regioselective post-synthetic modifications of nucleic acids are essential to studies of these molecules for science and applications. Here we report a facile universal approach by harnessing versatile phosphoramidation reactions to regioselectively incorporate alkynyl/azido groups into post-synthetic nucleic acids primed with phosphate at the 5' termini. With and without the presence of copper, the modified nucleic acids were subjected to azide-alkyne cycloaddition to afford various nucleic acid conjugates including a peptide-oligonucleotide conjugate (POC) with high yield. The POC was inoculated with human A549 cells and demonstrated excellent cell-penetrating ability despite cell deformation caused by a small amount of residual copper chelated to the POC. The combination of phosphoramidation and azide-alkyne cycloaddition reactions thus provides a universal regioselective strategy to post-synthetically modify nucleic acids. This study also explicated the toxicity of residual copper in synthesized bioconjugates destined for biological systems.

Effective and site-specific phosphoramidation reaction for universally labeling nucleic acids.

Here we report efficient and selective postsynthesis labeling strategies, based on an advanced phosphoramidation reaction, for nucleic acids of either synthetic or enzyme-catalyzed origin. The reactions provided phosphorimidazolide intermediates of DNA or RNA which, whether reacted in one pot (one-step) or purified (two-step), were directly or indirectly phosphoramidated with label molecules. The acquired fluorophore-labeled nucleic acids, prepared from the phosphoramidation reactions, demonstrated labeling efficacy by their F/N ratio values (number of fluorophores per molecule of nucleic acid) of 0.02-1.2 which are comparable or better than conventional postsynthesis fluorescent labeling methods for DNA and RNA. Yet, PCR and UV melting studies of the one-step phosphoramidation-prepared FITC-labeled DNA indicated that the reaction might facilitate nonspecific hybridization in nucleic acids. Intrinsic hybridization specificity of nucleic acids was, however, conserved in the two-step phosphoramidation reaction. The reaction of site-specific labeling nucleic acids at the 5'-end was supported by fluorescence quenching and UV melting studies of fluorophore-labeled DNA. The two-step phosphoramidation-based, effective, and site-specific labeling method has the potential to expedite critical research including visualization, quantification, structural determination, localization, and distribution of nucleic acids in vivo and in vitro.

The catalytic roles of P185 and T188 and substrate-binding loop flexibility in 3α-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni.

3α-Hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni reversibly catalyzes the oxidation of androsterone with NAD(+) to form androstanedione and NADH. Structurally the substrate-binding loop of the residues, T188-K208, is unresolved, while binding with NAD(+) causes the appearance of T188-P191 in the binary complex. This study determines the functional roles of the flexible substrate-binding loop in conformational changes and enzyme catalysis. A stopped-flow study reveals that the rate-limiting step in the reaction is the release of the NADH. The mutation at P185 in the hinge region and T188 in the loop causes a significant increase in the Kd value for NADH by fluorescence titration. A kinetic study of the mutants of P185A, P185G, T188A and T188S shows an increase in k(cat), K(androsterone) and K(iNAD) and equal primary isotope effects of (D)V and (D) (V/K). Therefore, these mutants increase the dissociation of the nucleotide cofactor, thereby increasing the rate of release of the product and producing the rate-limiting step in the hydride transfer. Simulated molecular modeling gives results that are consistent with the conformational change in the substrate-binding loop after NAD(+) binding. These results indicate that P185, T188 and the flexible substrate-binding loop are involved in binding with the nucleotide cofactor and with androsterone and are also involved in catalysis.

Advanced aqueous-phase phosphoramidation reactions for effectively synthesizing peptide-oligonucleotide conjugates trafficked into a human cell line.

Peptide-oligonucleotide conjugates (POCs) have held promise as effective therapeutic agents in treating microbial infections and human genetic diseases including cancers. In clinical applications, POCs are especially useful to circumvent cellular delivery and specificity problems of oligonucleotides. We previously reported that nucleic acid phosphoramidation reactions performed in aqueous solutions have the potential for facile POC synthesis. Here, we carried out further studies to significantly improve aqueous-phase two-step phosphoramidation reaction yield. Optimized reactions were employed to effectively synthesize POCs for delivery into human A549 cells. We achieved optimization of aqueous-phase two-step phosphoramidation reaction and improved reaction yield by (1) determining appropriate co-solutes and co-solute concentrations to acquire higher reaction yields, (2) exploring a different nucleophilicity of imidazole and its derivatives to stabilize essential nucleic acid phosphorimidazolide intermediates prior to POC formation, and (3) enhancing POC synthesis by increasing reactant nucleophilicity. The advanced two-step phosphoramidation reaction was exploited to effectively conjugate a well-studied cell penetrating peptide, the Tat(48-57) peptide, with oligonucleotides, bridged by either no linkers or a disulfide-containing linker, to have the corresponding POC yields of 47-75%. Phosphoramidation-synthesized POCs showed no cytotoxicity to human A549 cells at studied POC concentrations after 24 h inoculation and were successfully trafficked into the human A549 cell line as demonstrated by flow cytometry, fluorescent microscopy, and confocal laser scanning microscopy study. The current report provides insight into aqueous-phase phosphoramidation reactions, the knowledge of which was used to develop effective strategies for synthesizing POCs with crucial applications including therapeutic agents for medicine.

Gefitinib attenuates transforming growth factor-ß1-activated mitogen-activated protein kinases and mitogenesis in NRK-49F cells.

Transforming growth factor-ß (TGF-ß), TGF-ß receptor (TGF-ßR), and epidermal growth factor receptor (EGFR) are important in the pathogenesis of kidney fibrosis, a result of renal fibroblast activation. The EGFR kinase inhibitor gefitinib attenuates glomerular fibrosis in hypertensive rats whereas dominant-negative EGFR attenuates interstitial fibrosis in mouse with acute renal ischemia. Thus, we studied the effects and molecular mechanisms of gefitinib in TGF-ß1-induced mitogenesis and collagen production in normal rat kidney interstitial fibroblast (NRK-49F) cells. We found that TGF-ß1 increased cell mitogenesis. TGF-ß1 also time-dependently increased cyclin D1 protein expression. TGF-ß1 rapidly transactivated EGFR. SB431542 (a type I TGF-ßR kinase inhibitor) and SB203580 (a p38 kinase inhibitor) attenuated TGF-ß1-induced phosphorylation of Smad2/3 protein. SB431542 and gefitinib attenuated TGF-ß1-induced phosphorylation of ERK1/2 and p38 kinase. SB431542 and gefitinib also attenuated TGF-ß1-induced cyclin D1 protein expression. Moreover, SB431542, gefitinib, PD98059 (an ERK1/2 inhibitor), and SB203580 attenuated TGF-ß1-induced cell mitogenesis. Finally, SB431542 and gefitinib attenuated TGF-ß1-induced collagen production. We concluded that gefitinib attenuates TGF-ß1-induced cell mitogenesis via the EGFR-ERK1/2/p38 kinase pathway in NRK-49F cells. Moreover, gefitinib attenuates TGF-ß1-induced cyclin D1 protein expression and collagen production. Thus, gefitinib attenuates TGF-ß1-induced mitogenesis and collagen production in vitro.

Hemeoxygenase-1 expression in response to arecoline-induced oxidative stress in human umbilical vein endothelial cells.

BACKGROUND: Arecoline, the most abundant areca alkaloid, has been reported to stimulate reactive oxygen species (ROS) production in several cell types. Overproduction of ROS has been implicated in atherogenesis. Hemeoxygenase-1 (HO-1) has cytoprotective activities in vascular tissues. This study investigated the effect of arecoline on adhesion molecule expression and explored the role of HO-1 in this process. METHODS: Human umbilical vein endothelial cells (HUVECs) were treated with arecoline, then ROS levels and the expression of adhesion molecules and HO-1 were analyzed and potential signaling pathways investigated. RESULTS: After 2h of arecoline treatment, ROS production was stimulated and reached a maximum at 12h. Expression of the adhesion molecules ICAM and VCAM was also induced. Glutathione pretreatment completely blocked arecoline-stimulated ROS production and VCAM expression, but not ICAM expression. Arecoline also induced HO-1 expression and this effect was partly due by ROS stimulation. Inhibition of c-jun N-terminal kinase (JNK) by SP600125, p38 by SB 203580, or tyrosine kinase by genistein reduced arecoline-induced HO-1 expression. In contrast, inhibition of ERK (extracellular signal-related MAP kinase) by PD98059 had no effect. Transfection of HUVECs with the GFP/HO-1 gene, which resulted in a 5-fold increase in HO-1 activity, markedly, but not completely, inhibited the decrease in cell viability caused by arecoline. CONCLUSIONS: This study demonstrates that, in HUVECs, arecoline stimulates ROS production and ICAM and VCAM expression. HO-1 expression is also upregulated through the ROS, tyrosine kinase, and MAPK (JNK and p38) signaling pathways.

S100B protein expressions as an independent predictor of early relapse in UICC stages II and III colon cancer patients after curative resection.

BACKGROUND: S100 calcium-binding proteins such as S100B are elevated in primary malignant melanoma and are used as tumor markers for malignant melanoma and numerous other cancers. The purpose of this study was to identify the novel predictors of early relapse in UICC stages II and III colon cancer patients and thus to identify a subgroup of patients who are at high risk for postoperative early relapse. METHODS: Clinicopathological factors and S100B expression by immunohistochemical staining were retrospectively analyzed in 357 postoperative UICC stages II and III colon cancer patients to determine the predictors of early relapse. RESULTS: Of 357 patients, 114 patients developed postoperative relapse during the follow-up period. Among 114 relapsed colon cancer patients, postoperative early relapse and non-early relapse were found in 56 patients (49.1%) and 58 patients (50.9%), respectively. Multivariate Cox proportional hazards analysis revealed that the presence of vascular invasion (P = .025; hazard ratio [HR], 5.532; 95% confidence interval [95% CI], 1.985-14.729), high postoperative CEA levels (P = .019; HR, 6.845; 95% CI, 2.393-15.256), and S100B overexpression (P < .001; HR, 26.250; 95% CI, 7.463-96.804) were demonstrated to be independent predictors of postoperative early relapse. Furthermore, postoperative relapsed colon cancer patients with S100B overexpression were demonstrated to have significantly lower overall survival rates than those without S100B overexpression (P < .001). CONCLUSIONS: This study suggests that S100B protein expression is a crucial predictor of early relapse in UICC stages II and III postoperative colon cancer patients and thus could help to define patients with this tumor entity who would benefit from enhanced follow-up and therapeutic program(s).

Ling-Zhi polysaccharides potentiate cytotoxic effects of anticancer drugs against drug-resistant urothelial carcinoma cells.

The combined effects of ling-zhi polysaccharide fraction 3 (LZP-F3) and anticancer drugs (cisplatin and arsenic trioxide) were examined in three human urothelial carcinoma (UC) cells (parental, NTUB1; cisplatin-resistant, N/P(14); and arsenic-resistant, N/As(0.5)). MTT assay and median-effect analysis revealed that LZP-F3 could profoundly reverse the chemosensitivity of N/P(14) and N/As(0.5) to cisplatin and arsenic, respectively, in a dose-dependent manner, which involved activation of p38 and down-regulation of Akt and XPA. A dose of 10 mug/mL of LZP-F3 induced significant G1 arrest in N/P(14) and N/As(0.5) cells by flow cytometry, which may be mediated by the induction of p21(WAF1/CIP1). The combination of LZP-F3 and arsenic trioxide produced a significant synergistic growth inhibition of NTUB1 and N/As(0.5) cells. Similar results were also found in N/P(14) cells. These molecular events of combined effects involved significant and earlier induction of Fas, caspase 3 and 8 activation, Bax and Bad up-regulation, Bcl-2 and Bcl-x(L) down-regulatuion, and cytochrome c release.

Additive effects of C(2)-ceramide on paclitaxel-induced premature senescence of human lung cancer cells.

AIMS: the aims of the study are to investigate the additive effect of exogenous short-carbon chain phospholipids, C(2)-ceramide, on an anti-cancer drug paclitaxel (PTX)-induced senescence of human non-small cell lung cancer (NSCLC) cells deficient in functional p53 and p16, and to examine whether mitogen-activated protein kinase (MAPK) plays a role in ceramide-sensitized senescence of NSCLC cells. MAIN METHODS: to determine whether exogenous C(2)-ceramide renders lung cancer cells more sensitive to PTX treatment, techniques employing a flow cytometry-based cell cycle analysis and acidic ß-galactosidase staining for senescent cells were used. Furthermore, to elucidate the role of MAPK proteins in modulating senescence, assays for protein levels of selective MAPKs and Bcl-2 family members, and detection of transcriptional levels senescence-associated genes were used in the study. KEY FINDINGS: a sub-lethal dose of C(2)-ceramide sensitized the NSCLC H1299 cells to PTX treatment. The additive effects of C(2)-ceramide and PTX resulted in proliferative inhibition, G(2)-phase arrest of cell cycle, activation of p38 and eventually premature senescence. Importantly, neither p53, p21(waf1/cip1) nor p16(ink4) was shown to be involved in C(2)-ceramide-sensitized proliferative inhibition and senescence of H1299 cells by PTX in our study. SIGNIFICANCE: our study demonstrates that the short-carbon chain C(2)-ceramide can effectively sensitize PTX-induced senescence of H1299 cells via both p21(waf1/cip1)- and p16(ink4)-independent pathways.

DDR1 regulates the stabilization of cell surface E-cadherin and E-cadherin-mediated cell aggregation.

The stabilization of cell surface E-cadherin is important for the maintenance of apical junction complexes and epithelial polarity. Previously, we reported that discoidin domain receptor 1 (DDR1) forms a complex with E-cadherin at adhesive contacts; however, the regulatory role of DDR1 in the stabilization of cell surface E-cadherin and E-cadherin-mediated cell behaviors remained undefined. To gain insight into these questions, we utilized two stable clones depleted for DDR1 via the small interfering RNA (siRNA) technique, and we over-expressed DDR1 in MDCK cells. We performed Western blotting, immunofluorescence analysis, flow cytometry, and cell aggregation studies to investigate the effect of DDR1 on cell surface E-cadherin. The results showed that both DDR1/2 and E-cadherin use their extracellular domains to form DDR/E-cadherin complexes. Neither the depletion nor the over-expression of DDR1 changed the expression level of E-cadherin in MDCK cells. Collagen disrupted the formation of E-cadherin complexes and caused E-cadherin to accumulate in the cytoplasm; however, over-expression of DDR1 stabilized E-cadherin on the cell surface and decreased its cytoplasmic accumulation. Furthermore, independently of collagen stimulation, the depletion of DDR1 resulted in a decrease in the level of cell surface E-cadherin, which consequently caused its cytoplasmic accumulation and decreased E-cadherin-mediated cell aggregation. These results indicate that DDR1 can increase the stability of cell surface E-cadherin and promote MDCK cell aggregation, which may be mediated through the formation of DDR1/E-cadherin complexes. Overall, these findings have implications for the physiological roles of DDR1 in association with the maintenance of both the adhesion junction and epithelial polarity.

4beta-Hydroxywithanolide E from Physalis peruviana (golden berry) inhibits growth of human lung cancer cells through DNA damage, apoptosis and G2/M arrest.

BACKGROUND: The crude extract of the fruit bearing plant, Physalis peruviana (golden berry), demonstrated anti-hepatoma and anti-inflammatory activities. However, the cellular mechanism involved in this process is still unknown. METHODS: Herein, we isolated the main pure compound, 4beta-Hydroxywithanolide (4betaHWE) derived from golden berries, and investigated its antiproliferative effect on a human lung cancer cell line (H1299) using survival, cell cycle, and apoptosis analyses. An alkaline comet-nuclear extract (NE) assay was used to evaluate the DNA damage due to the drug. RESULTS: It was shown that DNA damage was significantly induced by 1, 5, and 10 microg/mL 4betaHWE for 2 h in a dose-dependent manner (p < 0.005). A trypan blue exclusion assay showed that the proliferation of cells was inhibited by 4betaHWE in both dose- and time-dependent manners (p < 0.05 and 0.001 for 24 and 48 h, respectively). The half maximal inhibitory concentrations (IC50) of 4betaHWE in H1299 cells for 24 and 48 h were 0.6 and 0.71 microg/mL, respectively, suggesting it could be a potential therapeutic agent against lung cancer. In a flow cytometric analysis, 4betaHWE produced cell cycle perturbation in the form of sub-G1 accumulation and slight arrest at the G2/M phase with 1 microg/mL for 12 and 24 h, respectively. Using flow cytometric and annexin V/propidium iodide immunofluorescence double-staining techniques, these phenomena were proven to be apoptosis and complete G2/M arrest for H1299 cells treated with 5 microg/mL for 24 h. CONCLUSIONS: In this study, we demonstrated that golden berry-derived 4betaHWE is a potential DNA-damaging and chemotherapeutic agent against lung cancer.