T47D Tumor Cells Research Articles

Study of nitrogen heterocycles as DNA/HSA binder, topoisomerase inhibitors and toxicological safety

Four new nitrogen-containing heterocyclic derivatives (acridine, quinoline, indole, pyridine) were synthesized and their biological properties were evaluated. The compounds showed affinity for DNA and HSA, with CAIC and CAAC displaying higher binding constants (Kb) of 9.54 × 104 and 1.06 × 106, respectively. The fluorescence quenching assay (Ksv) revealed suppression values ranging from 0.34 to 0.64 × 103 M−1 for ethidium bromide (EB) and 0.1 to 0.34 × 103 M−1 for acridine orange (AO). Molecular docking confirmed the competition of the derivatives with intercalation probes at the same binding site. At 10 μM concentrations, the derivatives inhibited topoisomerase IIα activity. In the antiproliferative assays, the compounds demonstrated activity against MCF-7 and T47-D tumor cells and nonhemolytic profile. Regarding toxicity, no acute effects were observed in the embryos. However, some compounds caused enzymatic and cardiac changes, particularly the CAIC, which increased SOD activity and altered heart rate compared to the control. These findings suggest potential antitumor action of the derivatives and indicate that substituting the acridine core with different cores does not interfere with their interaction and topoisomerase inhibition. Further investigations are required to assess possible toxicological effects, including reactive oxygen species generation.

Tissue factor-heparanase complex: intracellular nonhemostatic effects

BackgroundHeparanase, known to be involved in angiogenesis, cancer progression, and inflammation, was shown to form a complex with tissue factor (TF) via its procoagulant domain and to enhance the hemostatic system. ObjectivesTo reveal a potential role of heparanase procoagulant domain in nonhemostatic effects. MethodsEffects of peptides 16 and 16AC derived from the heparanase procoagulant domain, discovered by our group, were studied using the XTT proliferation assay, western blot analysis, and immunostaining in vitro and a mouse wound-healing model. ResultsProcoagulant peptides induced increased proliferation, release of heparanase, and upregulation of heparanase, TF, tissue factor pathway inhibitor (TFPI), and TFPI-2 in U87, T47D, and MCF-7 tumor cell lines and in endothelial cells. These results were reversed by a peptide derived from TFPI-2 that inhibited the heparanse procoagulant domain-TF complex. Thrombin had a similar effect on tumor cell proliferation and heparanase release, although the impact of thrombin on cell proliferation was mediated by the heparanase procoagulant domain. A mouse model of full-thickness skin incision exhibited higher levels of heparanase, TF, TFPI, and TFPI-2 in the healing skin, mainly in the blood vessel wall and lumen in animals injected with the procoagulant peptides compared to controls. The cells transfected to overexpress full-length TF or TF devoid of the cytoplasmic domain demonstrated that the procoagulant domain conveyed intracellular signaling via TF. ConclusionHeparanase procoagulant domain induces nonhemostatic effects via TF. The finding that TF serves as a receptor to heparanase supports the close direct relation between the hemostatic system and cancer progression.

Abstract LB194: Discovery and characterization of a mutant selective PI3KαH1047Xinhibitor with a best-in-class profile

Abstract Oncogenic PIK3CA mutations are found in 14% of all cancers and most frequently occur at amino acids E542/E545 and H1047, involving the helical and kinase domains, respectively (Zhang 2017). Alpelisib is a PI3Kα isoform-selective, orthosteric kinase inhibitor, having equivalent activity on wild-type (wt) and mutant (mt) forms and was approved to treat PI3Kα mt, HR+/HER2- breast cancer in combination with fulvestrant, nearly doubling progression-free survival (Andre 2019). Hyperglycemia with insulin resistance is an on-target result of wt PI3Kα inhibition, leading to frequent alpelisib dose reductions, interruptions, and discontinuations. H1047X is the most common PIK3CA alteration, found in ~14% of all breast cancer patients (Martínez-Sáez 2020). We considered that selectively targeting this mutant could improve patient outcomes by sparing metabolic dysfunction related to wt inhibition. We have validated this therapeutic concept preclinically with the discovery of ST-814, an allosteric, CNS-penetrant, mutant selective PI3KαH1047X inhibitor, displaying excellent drug-like properties. ST-814 was 14-fold selective for PI3KαH1047R vs PI3Kαwt in biochemical assays (IC50 11 vs 146 nM) with exquisite isoform and kinome selectivity. Cell-based activity and selectivity were characterized in T47D (PI3KαH1047R) and SKBR3 (PI3KαWT) tumor cells grown in 10% FBS, with 8-fold mut-selectivity observed in target engagement (pAKT EC50 38 vs 304 nM) and 11-fold selectivity in viability (GI50 153 vs 1683 nM). ST-814 has robust anti-tumor activity in PI3KαH1047X in xenografts, with efficacy similar or superior to alpelisib dosed at 50 mg/kg (Table 1). To achieve this level of mouse efficacy, alpelisib exposure exceeded that in patients >200% (mouse vs human AUC0-24, 74,000 vs 33,224 ng*hr/mL). As such, repeat dosing of alpelisib at 50 mg/kg, caused profound elevations in serum insulin 1 hr post-dose (*p<0.01), whereas ST-814 showed no significant difference (Table 1). The metabolic consequences of repeated alpelisib (50 mg/kg) and ST-814 (100 mg/kg) dosing was further characterized in an insulin tolerance test. Insulin-mediated glucose clearance (AUC0-4) was significantly impaired by alpelisib (p<0.01, one-way ANOVA), but not by ST-814. These data support the potential of molecules like ST-814 to improve outcomes in patients harboring H1047X mut tumors. A clinical candidate from this program is currently in IND enabling studies. Table 1. CAL33 H1047R Oral Sq Cell Carcinoma Xenograft Alpelisib TGI% 50 mg/kg ST-814 TGI% 100 mg/kg Alpelisib ΔInsulin ng/mL ST-814 ΔInsulin ng/mL 79% 82% 4.42* 0.40 Citation Format: Leonard Buckbinder, David J. St. Jean, Trang Tieu, Weixue Wang, Gregory Kryukov, Philip Jonsson, Jacob Alltucker, Samantha Manimala, Brendon Ladd, Angel Guzman-Perez, Darrin Stuart. Discovery and characterization of a mutant selective PI3KαH1047Xinhibitor with a best-in-class profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB194.

Photoprotective, biological activities and chemical composition of the non-toxic hydroalcoholic extract of Clarisia racemosa with cosmetic and pharmaceutical applications

Clarisia racemosa (guariúba), Moraceae, is a native tree of the Amazon region, with timber value in the local market and for export. Aiming to contribute to the biological study of the crude extract, we carried out its hydroalcoholic extraction and physicochemical characterization. The results showed that the wood had the following chemical composition: cellulose (40.1%), hemicellulose (18.34%), lignin (29.40%), extractives (10.7%), and ash (0.79%). The extract obtained showed a high content of phenolics and through liquid chromatography tests, it was possible to determine 17 compounds. Therefore, we evaluated its biological properties (antioxidant, antiglycant, in vitro and in vivo toxicity, antitumor, antimicrobial and photoprotective) to use it as a raw material in cosmetic and pharmaceutical formulations. The extract showed moderate antioxidant activity, it was also able to inhibit antiglycant reactions. And it showed no toxic effects in vitro and in vivo assays. Highlighting antiproliferative activity against MCF-7, T47D, DU-145, and Jurkat tumor cells. As reported in the literature, these models are still being used as a predictor of cytotoxicity. Regarding the in vitro antimicrobial assays, the extract showed activity against Staphylococcus aureus with a minimal inhibitory concentration similar to the oxacillin pattern. Finally, it was able to promote photoprotective activity in vitro at non-toxic concentrations. This study shows the potential of the hydroalcoholic extract of Clarisia racemosa to be used as a component in cosmetic and pharmaceutical formulations.

Design, synthesis, in vitro antiproliferative evaluation and GSK-3β kinase inhibition of a new series of pyrimidin-4-one based amide conjugates

A new series of novel amide conjugates of pyrimidin-4-one and aromatic/heteroaromatic /secondary cyclic amines has been synthesized and their in vitro antiproliferative activities against a panel of 60 human cancer cell lines of nine different cancer types were tested at NCI. Among the synthesized compounds, compound (4i) showed significant anti-proliferative activity. Compound (4i) displayed most potent activity against the breast tumor cell line T-47D and CNS tumor cell line SNB-75 exhibiting a growth of 1.93 % and 14.63 %, respectively. ADMET studies of the synthesized compounds were also performed and they were found to exhibit good drug like properties. Compound (4i) was found to exhibit potential inhibitory effect over GSK-3β with IC50 value of 71 nM. The molecular docking studies revealed that (4i) showed good binding affinity to GSK-3β and revealed multiple H-bonding and p-cation interactions with important amino acid residues on the receptor site. Compound (4i) may thus serve as a potential candidate for further development of novel anticancer therapeutics.

Targeted photodynamic therapy using reconstituted high-density lipoproteins as rhodamine transporters

Reconstituted high-density lipoprotein (rHDL) nanoparticles are excellent transporters of molecules and very useful for targeted therapy as they specifically recognize the scavenger receptor, class B1 (SR-B1) that is present on the surface of a wide range of tumor cells. However, they have rarely been employed to transport photosensitizers (PS) for photodynamic therapy (PDT). Rhodamine (R) compounds have been dismissed as useful PSs for PDT due to their low 1O2 production, excitation wavelengths with little tissue penetration, and poor selectivity for tumor cells. It was recently demonstrated that when irradiating at 532 nm or with Cerenkov radiation (CR) from a β-emitting radionuclide, R123, R6G, and RB undergo electron transfer reactions (type I reaction) with folic acid. R6G also produces type I reactions with O2. In this work, the photodynamic effects of the rHDL-R system were evaluated in vitro. rHDL nanoparticles loaded with R123, R6G, and RB were synthesized, and the PS was internalized into T47D tumor cells. When cells were irradiated with a 532-nm laser in the presence of an rHDL-R systems, a cytotoxic photodynamic effect was obtained in the order R6G > R123 > RB. In the presence of CR from a 177Lu source, cytotoxicity showed the order R6G > RB > R123. The higher cytotoxicity induced by R6G in both cases corresponds to higher cellular internalization and larger production of type I and II reactions. Thus, in this work, it is proposed that rHDL-R/177Lu system can be applied in theragnostics as a multimodal radiotherapy-PDT-imaging system (imaging by SPECT or Cerenkov) and in hypoxic solid tumors in which external radiation is not effective and 177Lu-CR acts as light source.

In vitro Activity of Novel Cannabinoids Derived from Tetrahydrocannabinolic Acid on Various Human Tumor Cell Lines

The in vitro study of tetracannabinolic acid (THCA) derivatives ALAM027 and ALAM108 was carried out on the following human tumor cells: T47D (breast, ductal carcinoma), PC-3 (prostate, adenocarcinoma), HT29 (colorectal carcinoma), Caco-2 (colon, adenocarcinoma), A549 (lung, carcinoma), U87MG (human glioblastoma) and U266B1 (multiple myeloma). The in vitro effects of THCA derivatives ALAM027 and ALAM108 on cell growth inhibition and IC50 values were measured using the CellTiter Glo assay. The ALAM027 compound showed good growth inhibition in all cell lines tested with the exception of U87MG cells. The ALAM108 compound also suppressed the growth of U87 MG cells but had little effect on T47D tumor cells. In vitro studies of THCA derivatives ALAM027 and ALAM108 showed antitumor activity in all cell lines tested. The difference in the activity of these compounds in relation to the T47D and U87MG tumor cells may be indicative of different functional mechanisms.

Electron transfer reactions in rhodamine: Potential use in photodynamic therapy

Rhodamine (R) compounds are photosensitizers with low production of 1O2, so these compounds have not been considered very often for photodynamic therapy (PDT). Rhodamine-6G (R6G) undergoes electron transfer reactions with folic acid (FA). This reaction converts this form of R into a potential photosensitizer (PS) for PDT. The occurrence of this reaction with other R or oxygen has not yet been proven. The low penetrability of visible light into the skin has led to the proposal of a PDT light source alternative to Cerenkov radiation (CR). This work evaluates and compares the occurrence of type-I transfer reactions of R6G, R123, and RB with FA and oxygen when irradiated at 532 nm. The potential use of these compounds as type-I photosensitizers for PDT was also evaluated in vitro by excitation with CR. R electron transfer reactions with FA and oxygen were monitored by emission fluorescence and ultraviolet/visible (UV/Vis) absorption of 2,6-dichlorophenol-indophenol (DCPIP) as a redox indicator, respectively. 177Lu and 18F were used as CR sources. The results showed that the three R compounds underwent type-I transfer reactions with FA in the order R123 > R6G > RB when irradiated at 532 nm. Excitation of R6G using CR was demonstrated. Only R6G showed a type-I reaction with O2. By irradiating T47D tumor cells with a 532 nm laser light, cell death occurred in the order R6G>R123>RB. The same trend was found when cells were irradiated with CR. The possibility of using the type-I reaction between R compounds and biomolecules provides a new perspective in the use of these dyes in PDT.

Co-culture of tumor spheroids and monocytes in a collagen matrix-embedded microfluidic device to study the migration of breast cancer cells

Recapitulating the tumor microenvironment is a major challenge in the development of in vitro tumor model for the study of cancer biology and therapeutic treatments. 3D multicellular tumor spheroids (MCTS) have been used as reliable models of mimicking in vivo solid tumors. Macrophages and extracellular matrix (ECM), regarded as two key factors of the tumor microenvironment, play significant roles in tumor progression and drug resistance. In order to investigate their effects on tumor cell migration, a microfluidic chip-based 3D breast cancer model was developed by co-culturing monodisperse MCTS with monocytes in 3D collagen matrix. A reversible bonding technique was employed for the fabrication of the microfluidic chip, which made it easier for MCTS formation and tailoring the MCTS co-culture conditions. When co-culturing monocytes with low invasive T47D spheroids or high invasive MD-MBA-231 spheroids, we found that T47D cells with the stimulation of macrophage colony-stimulating factor (M-CSF) and MD-MBA-231 cells could polarize monocytes into tumor-associated macrophages (TAMs). The increased stiffness via increasing collagen concentration decreased tumor cell migration, whereas the presence of TAMs enhanced the migration ability of cells. Moreover, M-CSF-activated TAMs promoted the migration of T47D tumor cells via the regulation of TGFβ1. Overall, this 3D co-culture microfluidic model may be useful for studying tumor progress and may offer a reliable and low-cost method for evaluation of drug efficiency.

Depsidones inhibit aromatase activity and tumor cell proliferation in a co-culture of human primary breast adipose fibroblasts and T47D breast tumor cells.

Naturally occurring depsidones from the marine fungus Aspergillus unguis are known to have substantial anti-cancer activity, but their mechanism of action remains elusive. The purpose of this study was to examine the anti-aromatase activity of two common depsidones, unguinol and aspergillusidone A, in a co-culture system of human primary breast adipose fibroblasts and hormonal responsive T47D breast tumor cells. Using this in vitro model it was shown that these depsidones inhibit the growth of T47D tumor cells most likely via inhibition of aromatase (CYP19) activity. The IC50 values of these depisidones were compared with the aromatase inhibitors letrozole and exemestane. Letrozole and exemestane had IC50 values of respectively, 0.19 and 0.14μM, while those for Unguinol and Aspergillusidone A were respectively, 9.7 and 7.3μM. Our results indicate that among the depsidones there maybe aromatase inhibitors with possible pharmacotherapeutical relevance.

Cytotoxic Effects of ZnO Nanoparticles on the Expression of ROS-Responsive Genes in the Human Cell Lines.

In the present investigation, engineered ZnO nanoparticles were tested for their induced oxidative stress in T47D tumor cell lines. The expressions of reactive oxygen species (ROS) related genes, glutathione S-transferase (GST) and catalase were quantified by real time-polymerase chain reaction (RT-PCR). In addition, qualitative analysis of GST was also performed at the cell level using molecular beacon (MB) technology. The tested nanoparticles were 20 nm in size, water-dispersible and treated on human breast tumor epithelial cell lines at 20, 40, 80 µg/ml concentration with 14, 28, 48 h incubation times. Nanoparticles induced expressions of ROS responsive genes at molecular and cellular level, produces consistent results with respect to different dosage and incubation time. The experiment showed that the expression of both GST and catalase genes were maximized at 28 h with 80 µg/ml concentration. However, the toxic effect of the monodisperse ZnO nanoparticles was not significant compared with control experiments, demonstrating its high potential in the applications of nanomedicines for a diagnostic and therapeutic tool.

Diterpenes and lignans from Viburnum odoratissimum var. odoratissimum

A new diterpenoid, dehydrovibsanin G (1), a new lignan, (+)-9′-O-senecioyllariciresinol (2), and six known compounds (3–8) were isolated from the branches and leaves of Viburnum odoratissimum var. odoratissimum. The structures of these compounds were elucidated by analysis of spectroscopic data. Both new compounds showed moderate inhibitory activity against human A431 and T47D tumor cell lines.

Synthesis, and QSAR analysis of anti-oncological active spiro-alkaloids.

QSAR study describes the anti-neoplastic spiro-alkaloids with relevant molecular descriptors using CODESSA III software. The dispiro[3H-indole-3,2'-pyrrolidine-3',3"-piperidines] 24-48 were synthesized via [3 + 2]-cycloaddition reaction of azomethine ylides, (generated in situ via decarboxylative condensation of isatins 21-23 with sarcosine) and 3E,5E-1-alkyl-3,5-bis(arylmethylidene)-4-piperidones 10-20. Some of the synthesized analogues exhibited promising antitumor properties against HELA (cervical), HEPG2 (liver), T-47D, MCF7 (breast), and HCT116 (colon) human tumor cell lines, demonstrating activity close to or even better than the standard Doxorubicin, based on in vitro Sulfo-Rhodamine-B bio-assay.

Abstract 3550: Inhibition of either LIN28A or ZCCHC11 (TUT4) provides distinct effects on the expression of the let-7 miRNA family and tumor cell proliferation

Abstract The LIN28A and LIN28B oncogenes are overexpressed in about 15% of human cancers, and they selectively block the expression of the tumor suppressor miRNA let-7 family, comprising twelve members (let-7a-1, -2, -3, let-7b, let-7c, let-7d, let-7e, let-7f-1, -2, let-7g, let-7i and miR-98) expressed from eight distinct loci. Upon binding to pre-let-7, LIN28A recruits ZCCHC11, a 3′ terminal uridylyl transferase, responsible for let-7 poly-uridylation and subsequent targeting of poly-uridylated let-7 miRNA for degradation. Expression of several let-7 miRNA family members was measured using quantitative RT-PCR in LIN28-positive or -negative tumor cell lines. In agreement with the inverse relationship between LIN28A/B and let-7 expression already observed by others (1), the lowest expression of let-7 miRNA was observed in LIN28A (e.g., IGROV-1 and T-47D) or LIN28B (e.g., NCI-H838, Hep-G2 and NCI-H1299) positive cell lines, and the degree of let-7 miRNA repression in LIN28A or LIN28B positive cell lines was particularly prominent for miR-98, let-7i and let-7b family members. Although the proliferation of LIN28B-positive tumor cell lines was sensitive to the 3 LIN28B siRNAs tested, the expression of LIN28A was not required for the proliferation and survival of LIN28A-expressing tumor cell lines, as demonstrated with one LIN28A siRNA that displayed an efficient knock-down at low concentrations with minimal impact on the proliferation of the T-47D tumor cell line. This LIN28A siRNA was further demonstrated to significantly upregulate the expression of several let-7 miRNAs such as miR-98 or let-7i, supporting an on-target modulation of the pathway. We also tested several siRNAs against ZCCHC11 uridylyl transferase, and found that the growth of either LIN28A- or LIN28B-positive tumor cell lines was inhibited by ZCCHC11 knockdown. However, ZCCHC11 protein knock-down was not able to restore let-7 miRNA expression, as it was seen for the LIN28A knock-down. These findings suggest that the role of ZCCHC11 in tumor cell lines might be more complex than just targeting poly-uridylated let-7 miRNA for degradation. Overall, our results seems to indicate that in LIN28A-positive tumor cell lines, LIN28A knock-down impacts let-7 miRNA expression, but has not a significant antiproliferative effect, whereas ZCCHC11 knock-down inhibits cell proliferation and this effect seems to be disconnected from let-7 miRNAs modulation. This lack of apparent effect on the expression of the miRNA let-7 expression might be related to the dual role of uridylyl transferases recently described for group II let-7 miRNAs, the mono- and poly-uridylation that leads to let-7 biogenesis and let-7 degradation respectively (2). 1- Viswanathan SR, Powers JT, Einhorn W, et al. Nat Genet 2009; 41: 843-848. 2- Heo I, Ha M, Lim J et al. Cell 2012; 151: 521-532. Citation Format: Laurent VIDARD, Claire MARIET, Eric BOITIER, Véronique SIERRA, Elisabeth CAVROIS, Carlos GARCIA-ECHEVERRIA, Hélène GOULAOUIC. Inhibition of either LIN28A or ZCCHC11 (TUT4) provides distinct effects on the expression of the let-7 miRNA family and tumor cell proliferation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3550. doi:10.1158/1538-7445.AM2014-3550

Metabolomic characterization of human breast T47D tumor cell phospholibids by UPLC-MS/MS and GC-MS

Metabolomic characterization of human breast T47D tumor cell phospholibids by UPLC-MS/MS and GC-MS

Metabolomic characterization of human breast T47D tumor cell phospholipids by UPLC-MS/MS and GC-MS

Metabolomic characterization of human breast T47D tumor cell phospholipids by UPLC-MS/MS and GC-MS

Abstract P5-05-07: Promestriene effects on estrogen-sensitive breast cancer cell proliferation in vitro

Abstract Introduction: Promestriene (3-propyl ethyl, 17B-methyl estradiol) is a synthetic estrogen analogue with potential as a topical treatment for vaginal atrophy caused by estrogen deprivation. Topical Promestriene is minimally absorbed with chronic use and has been reported to significantly improve the symptoms of vaginal atrophy. However, to be considered for use for these symptoms in estrogen responsive post-menopausal breast cancer survivors on an aromatase inhibitor, Promestriene would need to show none of the cyto-proliferative properties of estrogen. Methods: We conducted in vitro testing of estrogen responsive breast cancer cell line MCF-7, T-47D, and BT-474 for dose-dependent, estrogen-like functional responses to Promestriene in both estrogen-rich and estrogen-deprived conditions. As assay controls, cultures treated with Fulvestrant, Testosterone, Progesterone and Estradiol were analyzed in parallel. Analysis: Dose dependent Estrogen-like responses in the cell lines were measured as 1) proliferation using flow cytometric analysis of CSFE fluorescence, 2) estrogen receptor expression by flow cytometry, and 3) GREB 1 RNA expression using TaqMan real time PCR quantitative analysis. All cultures were grown to quiescence in RPMI medium supplemented with 10% fetal calf serum. To induce estrogen deprivation, half of the cultures were then held 3 days in media pre-treated and containing anti-estrogen specific antibodies to sequester any residual estrogen from the culture. Data for each culture treatment were analyzed using linear regression analysis and compared using multifactorial or pairwise analyses to determine the significance of estrogen-like responses relative to the estradiol and untreated controls. Results: In estrogen sufficient cultures, Promestriene exhibited no cell proliferative properties on MCF-7, BT-474, or T47-D tumor cells, even at very high concentrations. However, when estrogen in the media was sequestered and culture allowed to quiesce without estrogen receptor stimulation, low dose Promestriene effects on cell proliferation in MCF-7 estrogen responsive cells were not significantly different from low dose estradiol. Low dose Promestriene (2-10pg/ml), unlike comparable doses of estradiol, did not stimulate GREB1 expression in MCF-7 cells. In contrast, T-47D and BT-474 cell lines proliferated and increased GREB- expression in response Promestriene or Fulvestrant treatments. Conclusions: The potential estrogen-like properties of Promestriene to stimulate the growth of estrogen receptor responsive breast cancer cell lines, especially in estrogen-deprived conditions, suggest caution when prescribed for vaginal atrophy in post-menopausal breast cancer survivors on an aromatase inhibitor. Its ability to activate growth and gene expression in ER- breast cancer cells warrants further study. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-05-07.

Asteropsiside A and other asterosaponins from the starfish Asteropsis carinifera

Three asterosaponins were isolated from the tropical starfish Asteropsis carinifera: a new one, asteropsiside A, and two known ones, regularoside A and thornasteroside A. The structure of the new compound was established using 2D NMR spectroscopy and ESI mass spectrometry as the sodium salt of 3-O-sulfonato-(20E)-6-O-{β-d-fucopyranosyl-(1→2)-β-d-galactopyranosyl-(1→4)-[β-d-quinovopyranosyl-(1→2)]-β-d-xylopyranosyl-(1→3)-β-d-quinovopyranosyl}-3β,6α-dihydroxy-5α-cholesta-9(11),20(22)-dien-23-one. Regularoside A and thornasteroside A were shown to display the ability to inhibit the growth of the T-47D and RPMI-7951 tumor cell colonies in vitro.

Targeted Delivery of Methotrexate to Tumor Cells Using Biotin Functionalized Methotrexate-Human Serum Albumin Conjugated Nanoparticles

Systemic toxicity following cancer chemotherapy is an important concern. Targeted drug delivery systems could reduce the toxicity of anticancer drugs. Vitamins have been considered as targeting moieties in novel cancer treatment strategies. In this study, biotin was attached to nanoparticles of conjugated methotrexate-human serum albumin. Biotin functionalized methotrexate-human serum albumin with three different amounts of biotin attached to the nanoparticles were prepared. It was shown that the cytotoxicity of biotin functionalized nanoparticles on T47D and HeLa tumor cells was significantly higher than that of free methotrexate and non-functionalized nanoparticles. The cytotoxicity of biotin functionalized nanoparticles further increased when the number of biotin molecules attached on the surface of nanoparticles increased. The uptake of FITC labeled biotin functionalized nanoparticles by T47D and HeLa cells measured by flow cytometry, was also higher than that of non-functionalized nanoparticles. It can be concluded that the biotin functionalized methotrexate-human serum albumin conjugated nanoparticles could be used as a potent drug for specific delivery of methotrexate to tumors.

Molecular Recognition of the Tes LIM2–3 Domains by the Actin-related Protein Arp7A

Actin-related proteins (Arps) are a highly conserved family of proteins that have extensive sequence and structural similarity to actin. All characterized Arps are components of large multimeric complexes associated with chromatin or the cytoskeleton. In addition, the human genome encodes five conserved but largely uncharacterized “orphan” Arps, which appear to be mostly testis-specific. Here we show that Arp7A, which has 43% sequence identity with β-actin, forms a complex with the cytoskeletal proteins Tes and Mena in the subacrosomal layer of round spermatids. The N-terminal 65-residue extension to the actin-like fold of Arp7A interacts directly with Tes. The crystal structure of the 1–65Arp7A·LIM2–3Tes·EVH1Mena complex reveals that residues 28–49 of Arp7A contact the LIM2–3 domains of Tes. Two alanine residues from Arp7A that occupy equivalent apolar pockets in both LIM domains as well as an intervening GPAK linker that binds the LIM2–3 junction are critical for the Arp7A-Tes interaction. Equivalent occupied apolar pockets are also seen in the tandem LIM domain structures of LMO4 and Lhx3 bound to unrelated ligands. Our results indicate that apolar pocket interactions are a common feature of tandem LIM domain interactions, but ligand specificity is principally determined by the linker sequence.