Synthesis, in silico, in vitro evaluation of furanyl- and thiophenyl-3-phenyl-1H-indole-2-carbohydrazide derivatives as tubulin inhibitors and anticancer agents.

Twenty-one new indole derivatives comprising of seven furanyl-3-phenyl-1H-indole-carbohydrazide derivatives and fourteen thiophenyl-3-phenyl-1H-indole-carbohydrazide derivatives were synthesised and biologically evaluated for their microtubule-destabilising effects, and antiproliferative activities against the National Cancer Institute 60 (NCI60) human cancer cell line panel. Among the derivatives, 6i showed the best cytotoxic activity exhibiting selectivity for COLO 205 colon cancer (LC50 = 71 nM), SK-MEL-5 melanoma cells (LC50 = 75 nM), and MDA-MB-435 (LC50 = 259 nM). Derivative 6j showed the strongest microtubule-destabilising effect. Both 6i and 6j were able to induce G2/M cell cycle arrest and apoptosis in MDA-MB-231 triple-negative breast cancer cells. Molecular docking simulation results suggested that these derivatives inhibit tubulin by binding at the colchicine site. The calculated molecular descriptors showed that the most potent derivatives have acceptable pharmacokinetic profiles and are favourable for oral drug administration.

Anthraquinones from the roots of Morinda scabrida Craib exhibit antiproliferative activity against A549 lung cancer cells and antitubulin polymerization.

Six anthraquinones were isolated from Morinda scabrida Craib, an unexplored species of Morinda found in the tropical forest of Thailand. All six anthraquinones showed cytotoxicity against A549 lung cancer cells, with the most active compound, nordamnacanthal (MS01), exhibiting the IC50 value of 16.3 ± 2.5 µM. The cytotoxic effect was dose-dependent and led to cell morphological changes characteristic of apoptosis. In addition, flow cytometric analysis showed dose-dependent apoptosis induction and the G2/M phase cell cycle arrest, which was in agreement with the tubulin polymerization inhibitory activity of MS01. Molecular docking analysis illustrated the binding between MS01 and the α/ß-tubulin heterodimer at the colchicine binding site, and UV-visible absorption spectroscopy revealed the DNA binding capacity of MS01.

The amide derivative of anticopalic acid induces non-apoptotic cell death in triple-negative breast cancer cells by inhibiting FAK activation.

Anticopalic acid (ACP), a labdane type diterpenoid obtained from Kaempferia elegans rhizomes, together with 21 semi-synthetic derivatives, were evaluated for their cancer cytotoxic activity. Most derivatives displayed higher cytotoxic activity than the parent compound ACP in a panel of nine cancer cell lines. Among the tested compounds, the amide 4p showed the highest cytotoxic activity toward leukemia cell lines, HL-60 and MOLT-3, with IC50 values of 6.81 ± 1.99 and 3.72 ± 0.26 µM, respectively. More interestingly, the amide derivative 4l exhibited cytotoxic activity with an IC50 of 13.73 ± 0.04 µM against the MDA-MB-231 triple-negative breast cancer cell line, which is the most aggressive type of breast cancer. Mechanistic studies revealed that 4l induced cell death in MDA-MB-231 cells through non-apoptotic regulated cell death. In addition, western blot analysis showed that compound 4l decreased the phosphorylation of FAK protein in a concentration-dependent manner. Molecular docking simulations elucidated that compound 4l could potentially inhibit FAK activation by binding to a pocket of FAK kinase domain. The data suggested that compound 4l could be a potential FAK inhibitor for treating triple-negative breast cancer and worth being further investigated.

Turmeric Herb Extract-Incorporated Biopolymer Dressings with Beneficial Antibacterial, Antioxidant and Anti-Inflammatory Properties for Wound Healing.

Bacterial infection and inflammation caused by excess oxidative stress are serious challenges in chronic wound healing. The aim of this work is to investigate a wound dressing based on natural- and biowaste-derived biopolymers loaded with an herb extract that demonstrates antibacterial, antioxidant, and anti-inflammatory activities without using additional synthetic drugs. Turmeric extract-loaded carboxymethyl cellulose/silk sericin dressings were produced by esterification crosslinking with citric acid followed by freeze-drying to achieve an interconnected porous structure, sufficient mechanical properties, and hydrogel formation in situ in contact with an aqueous solution. The dressings exhibited inhibitory effects on the growth of bacterial strains that were related to the controlled release of the turmeric extract. The dressings provided antioxidant activity as a result of the radical scavenging effect on DPPH, ABTS, and FRAP radicals. To confirm their anti-inflammatory effects, the inhibition of nitric oxide production in activated RAW 264.7 macrophages was investigated. The findings suggested that the dressings could be a potential candidate for wound healing.

PLGA-Lipid Hybrid Nanoparticles for Overcoming Paclitaxel Tolerance in Anoikis-Resistant Lung Cancer Cells.

Drug resistance and metastasis are two major obstacles to cancer chemotherapy. During metastasis, cancer cells can survive as floating cells in the blood or lymphatic circulatory system, due to the acquisition of resistance to anoikis-a programmed cell death activated by loss of extracellular matrix attachment. The anoikis-resistant lung cancer cells also develop drug resistance. In this study, paclitaxel-encapsulated PLGA-lipid hybrid nanoparticles (PLHNPs) were formulated by nanoprecipitation combined with self-assembly. The paclitaxel-PLHNPs had an average particle size of 103.0 ± 1.6 nm and a zeta potential value of -52.9 mV with the monodisperse distribution. Cytotoxicity of the nanoparticles was evaluated in A549 human lung cancer cells cultivated as floating cells under non-adherent conditions, compared with A549 attached cells. The floating cells exhibited anoikis resistance as shown by a lack of caspase-3 activation, in contrast to floating normal epithelial cells. Paclitaxel tolerance was evident in floating cells which had an IC50 value of 418.56 nM, compared to an IC50 value of 7.88 nM for attached cells. Paclitaxel-PLHNPs significantly reduced the IC50 values in both attached cells (IC50 value of 0.11 nM, 71.6-fold decrease) and floating cells (IC50 value of 1.13 nM, 370.4-fold decrease). This report demonstrated the potential of PLHNPs to improve the efficacy of the chemotherapeutic drug paclitaxel, for eradicating anoikis-resistant lung cancer cells during metastasis.

Acetogenins from the stems of Uvaria micrantha showing antiproliferative effects on HepG2 liver cancer cells.

Five mono-tetrahydrofuran acetogenins: uvamicranins A-E and three known mono-tetrahydrofuran acetogenins; reticulatacin, calamistrin A, and uvarigrin, were isolated from the stems of Uvaria micrantha (Annonaceae). Their structures were elucidated by 2D NMR and high-resolution mass spectral analysis. The absolute configurations of uvamicranins A and B were determined by modified Mosher's method. Evaluation of antiproliferative activity of the isolated compounds showed that they were more potent towards the human hepatocellular carcinoma cell line HepG2, compared to the five other tested cell lines. Among the tested compounds, uvamicranin B (UvB) and uvarigrin (Uv) possessed strong antiproliferative activity with IC50 values of 2.89 ± 0.71 µM and 0.37 ± 0.06 µM, respectively. The antiproliferative mechanism of UvB and Uv, was investigated in HepG2 cell line showing that both compounds marginally induced apoptotic cell death, but exhibited cytostatic effect through induction of cell cycle arrest at the G2/M phase.

Biphasic Dose-Dependent G0/G1 and G2/M Cell-Cycle Arrest by Synthetic 2,3-Arylpyridylindole Derivatives in A549 Lung Cancer Cells.

A collection of 2,3-arylpyridylindole derivatives were synthesized via the Larock heteroannulation and evaluated for their in vitro cytotoxic activity against A549 human lung cancer cells. Two derivatives expressed good cytotoxicity with IC50 values of 1.18±0.25 µM and 0.87±0.10 µM and inhibited tubulin polymerization in vitro, with molecular docking studies suggesting the binding modes of the compounds in the colchicine binding site. Both derivatives have biphasic cell cycle arrest effects depending on their concentrations. At a lower concentration (0.5 µM), the two compounds induced G0/G1 cell cycle arrest by activating the JNK/p53/p21 pathway. At a higher concentration (2.0 µM), the two derivatives arrested the cell cycle at the G2/M phase via Akt signaling and inhibition of tubulin polymerization. Additional cytotoxic mechanisms of the two compounds involved the decreased expression of Bcl-2 and Mcl-1 antiapoptotic proteins through inhibition of the STAT3 and Akt signaling pathways.

JAK2/STAT3­mediated dose­dependent cytostatic and cytotoxic effects of sesquiterpene lactones from Gymnanthemum extensum on A549 human lung carcinoma cells.

Due to drug resistance and disease recurrence, lung cancer remains one of the primary cancer­related causes of death in both men and women worldwide. In addition, lung cancer is clinically silent and thus most patients are at an advanced stage at the time of diagnosis. The limited efficiency of current conventional chemotherapies necessitates the search for novel effective anticancer agents. The present study demonstrated the anti­proliferative effect and apoptosis­inducing activity of three sesquiterpene lactones isolated from Gymnanthemum extensum, vernodalin (VDa), vernolepin (VLe) and vernolide (VLi), on A549 human lung cancer cells. Treatment with sub­cytotoxic doses (cell viability remaining >75%) of VDa, VLe and VLi, arrested progression of the A549 cell cycle at the G0/G1 phase, while cytotoxic doses of the three compounds induced G2/M phase arrest and apoptosis. Mechanistic studies revealed that VDa, VLe and VLi may exert their anti­tumor activity through the JAK2/STAT3 pathway. Molecular docking analysis confirmed that VDa, VLe and VLi formed hydrogen bonds with the FERM domain of JAK2 protein. Overall, the present study highlighted the potential therapeutic value of VDa, VLe and VLi to be further developed as anticancer agents for the treatment of lung cancer.

Synthesis of Isocryptolepine-Triazole Adducts and Evaluation of Their Cytotoxic Activity.

Eighteen hybrid compounds between 8-bromo-2-fluoro-isocryptolepine (4) and 1,2,3-triazole were synthesized via azide rearrangement-annulation reaction. Compound 4 underwent regioselective N-propargylation and click reaction to form 8-bromo-2-fluoro-isocryptolepine-triazole hybrids 11 which were evaluated for cytotoxic activity. Compound 11 c containing 1-anisyltriazole was the most effective in inhibiting HepG2, HuCCA-1 and A549 cell lines (IC50 values of 1.65-3.07 µM) while compounds 11 a (1-phenyltriazole), 11 j (1-para-CF3 -benzyltriazole) and 11 l (1-meta-Cl-benzyltriazole) were potent inhibitors of HuCCA-1, HepG2 and A549 cell lines, respectively. Moreover, 11 l showed the lowest cytotoxicity to normal human kidney cell line. Compounds 11 c and 11 l provided improvement of cytotoxic activity over 4. Compounds 4, 11 c and 11 l were selected to investigate their mechanisms of action. The results showed that 4 could induce G2/M cell cycle arrest and was involved in the upregulation of p53 and p21 proteins. However, the mechanisms of growth inhibition by 11 c and 11 l were associated with G0/G1 cell cycle arrest and mediated by induction of oxidative stress.

Identification of new 3-phenyl-1H-indole-2-carbohydrazide derivatives and their structure-activity relationships as potent tubulin inhibitors and anticancer agents: A combined in silico, in vitro and synthetic study.

Virtual screening of commercially available molecular entities by using CDRUG, structure-based virtual screening, and similarity identified eight new derivatives of 3-phenyl-1H-indole-2-carbohydrazide with anti-proliferative activities. The molecules were tested experimentally for inhibition of tubulin polymerisation, which revealed furan-3-ylmethylene-3-phenyl-1H-indole-2-carbohydrazide (27a) as the most potent candidate. Molecule 27a was able to induce G2/M phase arrest in A549 cell line, similar to other tubulin inhibitors. Synthetic modifications of 27a were focussed on small substitutions on the furan ring, halogenation at R1 position and alteration of furyl connectivity. Derivatives 27b, 27d and 27i exhibited the strongest tubulin inhibition activities and were comparable to 27a. Bromine substitution at R1 position showed most prominent anticancer activities; derivatives 27b-27d displayed the strongest activities against HuCCA-1 cell line and were more potent than doxorubicin and the parent molecule 27a with IC50 values <0.5 µM. Notably, 27b with a 5-methoxy substitution on furan displayed the strongest activity against HepG2 cell line (IC50 = 0.34 µM), while 27d displayed stronger activity against A549 cell line (IC50 = 0.43 µM) compared to doxorubicin and 27a. Fluorine substitutions at the R1 position tended to show more modest anti-tubulin and anticancer activities, and change of 2-furyl to 3-furyl was tolerable. The new derivatives, thiophenyl 26, displayed the strongest activity against A549 cell line (IC50 = 0.19 µM), while 1-phenylethylidene 21b and 21c exhibited more modest anticancer activities with unclear mechanisms of action; 26 and 21c demonstrated G2/M phase arrest, but showed weak tubulin inhibitory properties. Molecular docking suggests the series inhibit tubulin at the colchicine site, in agreement with the experimental findings. The calculated molecular descriptors indicated that the molecules obey Lipinski's rule which suggests the molecules are drug-like structures.

Molecular dynamics, MM/PBSA and in vitro validation of a novel quinazoline-based EGFR tyrosine kinase inhibitor identified using structure-based in silico screening.

EGFR-TK has been a target strongly associated with the development of NSCLCs. A structure-based virtual screening campaign was launched against EGFR-TK by virtual screening a 3D library of 167 commercially available small molecules downloaded from ChemBridge Corporation. The virtual screen identified 12 virtual hit molecules, which were biologically evaluated against an EGFR-TK inhibitor-sensitive NSCLC cell line, A549. A quinazoline-based molecule 1, was most active and displayed ∼58% cytotoxicity at 20 µM single dose. The mode of cell death suggests molecule 1 induced apoptosis, which is characteristic of EGFR-TK pathway inhibition. A 50 ns MD simulation was conducted on three different systems: free EGFR-TK, molecule 1 complexed to EGFR-TK, and the positive control, lapatinib, complexed to EGFR-TK. The MD simulations showed increase in stabilisation of the EGFR-TK structure for the complexed systems, i.e., lower RMSDs and RMSFs for complexed EGFR-TK structures compared to the free EGFR-TK system. The binding affinities were estimated using MM/PBSA in the last 10 ns of the MD simulation that revealed comparable binding free energies between molecule 1 and lapatinib, ΔGbind = -25.0 and -23.9 kcal/mol, respectively. Per residue binding free energy decomposition studies revealed non-polar interactions contributed mostly to the binding free energies. Residues Leu718, Arg841 and Phe856 were predicted to contribute most to the binding free energies for molecule 1.

Overcoming the diverse mechanisms of multidrug resistance in lung cancer cells by photodynamic therapy using pTHPP-loaded PLGA-lipid hybrid nanoparticles.

Multidrug resistance (MDR) and the spread of cancer cells (metastasis) are major causes leading to failure of cancer treatment. MDR can develop in two main ways, with differences in their mechanisms for drug resistance, first drug-selected MDR developing after chemotherapeutic treatment, and metastasis-associated MDR acquired by cellular adaptation to microenvironmental changes during metastasis. This study aims to use a nanoparticle-mediated photodynamic therapy (NPs/PDT) approach to overcome both types of MDR. A photosensitizer, 5,10,15,20-Tetrakis(4-hydroxy-phenyl)-21H,23H-porphine (pTHPP) was loaded into poly(D,L-lactide-co-glycolide) (PLGA)-lipid hybrid nanoparticles. The photocytotoxic effect of the nanoparticles was evaluated using two different MDR models established from one cell line, A549 human lung adenocarcinoma, including (1) A549RT-eto, a MDR cell line derived from A549 cells by drug-selection, and (2) detachment-induced MDR acquired by A549 cells when cultured as floating cells under non-adherent conditions, which mimic metastasizing cancer cells in the blood/lymphatic circulation. In the drug-selected MDR model, A549RT-eto cells displayed 17.4- and 1.8-fold resistance to Etoposide and Paclitaxel, respectively, compared to the A549 parental cells. In contrast to treatment with anticancer drugs, NPs/PDT with pTHPP-loaded nanoparticles resulted in equal photocytotoxic effect in A549RT-eto and parental cells. Intracellular pTHPP accumulation and light-induced superoxide anion generation were observed at similar levels in the two cell lines. The NPs/PDT killed A549RT-eto and parental cells through apoptosis as revealed by flow cytometry. In the metastasis-associated MDR model, A549 floating cells exhibited resistance to Etoposide (11.6-fold) and Paclitaxel (57.8-fold) compared to A549 attached cells, but the floating cells failed to show resistance against the photocytotoxic effect of the NPs/PDT. The MDR overcoming activity of NPs/PDT is mainly due to delivery ability of the PLGA-lipid hybrid nanoparticles. In conclusion, this work suggests that PLGA-lipid hybrid nanoparticles have potential in delivering photosensitizer or chemotherapeutic drug for treating both drug-selected and metastasis-associated MDR lung cancer cells.

Mycophenolic acid is a drug with the potential to be repurposed for suppressing tumor growth and metastasis in osteosarcoma treatment.

Our previous review of proteomics data showed that in osteosarcoma, some overexpressed proteins were targets of FDA-approved immunosuppressive and anti-arrhythmic drugs, including mycophenolate mofetil (MMF), ribavirin, leflunomide, azathioprine and digoxin. Here, these drugs were screened for growth inhibitory effects in human osteosarcoma cell lines, including MNNG/HOS, U2OS, SaOS-2, MG-63 and 143B cells. Only mycophenolic acid (MPA), an active metabolite of MMF, efficiently inhibited osteosarcoma cell growth with IC50 values of 0.46-7.3 µM; these values are in the therapeutic range for organ transplant patients. At a therapeutic dose (10 µM), MPA significantly inhibited colony formation, caused cell cycle arrest in the S phase, and induced apoptosis. Moreover, the in vitro invasion of osteosarcoma cells was reduced by MPA by inhibiting cell migration capability. The in vivo antitumor effect of MMF was determined in nude mice harboring 143B cell xenografts. Daily oral administration of 200 mg/kg/day MMF for 2 weeks significantly suppressed tumor growth in treated mice, achieving 57.4 ± 11.1% tumor growth inhibition. Compared with the vehicle group, the MMF group treated with 50-200 mg/kg/day for 3 weeks had a significant reduction in the number of lung metastatic nodules in a tail vein-lung metastasis model of 143B cells. MMF doses of 50, 100 and 200 mg/kg/day are approximately equivalent to the non-toxic doses of 0.25, 0.5 and 1 g/day in humans, respectively. These findings indicate that MPA/MMF can effectively control osteosarcoma tumor growth and metastasis. Thus, the potential to repurpose MPA/MMF for use in osteosarcoma chemotherapy is of great interest.

A synthetic 2,3-diarylindole induces microtubule destabilization and G2/M cell cycle arrest in lung cancer cells.

The anticancer potential of a synthetic 2,3-diarylindole (PCNT13) has been demonstrated in A549 lung cancer cells by inducing both apoptosis and autophagic cell death. In this report, we designed to connect a fluorophore to the compound via a hydrophilic linker for monitoring intracellular localization. The best position for linker attachment was identified from cytotoxicity and effect on cell morphology of newly synthesized PCNT13 derivatives bearing hydrophilic linker. Cytotoxicity and effect on cell morphology related to the parental compound were used to identify the optimum position for linker attachment in the PCNT13 chemical structure. The fluorophore-PCNT13 conjugate was found to localize in the cytoplasm. Microtubules were found to be one of the cytosolic target proteins of PCNT13, as the compound could inhibit tubulin polymerization in vitro. A molecular docking study revealed that PCNT13 binds at the colchicine binding site on the α/ß-tubulin heterodimer. The effect of PCNT13 on microtubule dynamics caused cell cycle arrest in the G2/M phase as analyzed by flow cytometric analysis.

Vernodalidimer L, a sesquiterpene lactone dimer from Vernonia extensa and anti-tumor effects of vernodalin, vernolepin, and vernolide on HepG2 liver cancer cells.

Vernonia extensa, known as "Phim Phai Lin" in Thai, is distributed in most regions of Thailand. The plant has been used in Ayurveda and traditionally used to treat malaria and cancer, and possesses several sesquiterpene lactones. This study aimed to investigate and identify the active constituents by bioactivity-based analysis, as well as to evaluate the cytotoxic activity of V. extensa by MTT or XTT assays in ten cancer cell lines (Liver HepG2 and S102; Bile duct HuCCA-1; Leukemia HL-60 and MOLT-3; Lung A549 and H69AR; Breast MDA-MB-231 and T47D; Cervical HeLa). Bioactivity-guided fractionation and semi-preparative HPLC purification were used to separate the bioactive constituents. Apoptosis-inducing activity and cell cycle inhibitory effect of selected active compounds were determined on HepG2 cells by flow cytometric analysis. Bioactivity-guided fractionation of the CH2Cl2 extract and chemical investigation of the cytotoxic fractions led to the isolation of a new sesquiterpenoid pseudo-dimer named vernodalidimer L, together with eight known sesquiterpenoids from the aerial part of V. extensa. The structures of the isolates were elucidated based on spectroscopic analysis, including 1D and 2D NMR and HRMS. Vernolide has potent broad-spectrum cytotoxicity with IC50 values in the range of 0.91-13.84 µM, against all ten cancer cell lines. The annexin-V flow cytometric analysis showed that vernodalin, vernolepin, and vernolide induced apoptosis on HepG2 cells in a dose dependent manner and these effects correlated with G2/M phase cell cycle arrest. Our results indicated that vernodalin, vernolepin, and vernolide have potential to be used as lead compounds in the development of a therapeutic natural product for treatment of liver cancer.

Perfluorooctanoic Acid Enhances Invasion of Follicular Thyroid Carcinoma Cells Through NF-κB and Matrix Metalloproteinase-2 Activation.

BACKGROUND/AIM: Perfluorooctanoic acid (PFOA) is one of the most common perfluorinated compounds widely used in several applications. Due to its persistence in the environment, PFOA has been associated with various diseases, including cancer. This study explored the effects of PFOA on follicular thyroid carcinoma cells (FTC133). MATERIALS AND METHODS: Cell invasion, migration, adhesion and activity of matrix metalloproteinase-2 (MMP-2) were investigated using Transwell assays, adhesion assay and gelatin zymography, respectively. The underlying mechanism involved in the effects observed was evaluated by immunoblot analyses. RESULTS: Treatment with PFOA did not affect cell migration, but enhanced cell invasion, adhesion and activity of MMP-2 in FTC133 cells. PFOA selectively enhanced the phosphorylation of nuclear factor kappa B (NF-κB) p65, as well as induced NF-κB nuclear translocation. Treatment with a NF-κB inhibitor (BAY 11-7085) was able to reverse PFOA-induced cell invasiveness. CONCLUSION: PFOA promotes invasiveness of FTC133 cells mediated through the activation of NF-κB signaling.

Shikonin Suppresses Lymphangiogenesis via NF-κB/HIF-1α Axis Inhibition.

Lymphangiogenesis, the formation of lymphatic vessels from preexisting ones, promotes cancer growth and metastasis. Finding natural compounds with anti-lymphangiogenic activity will be useful for preventive treatment of lymphatic metastasis. Shikonin, an ingredient of a traditional Japanese and Chinese medicinal herb Lithospermum erythrorhizon, has been widely used in several pharmaceutical and cosmetic preparations, as well as in food colorants. Shikonin has been reported to inhibit lymphangiogenesis in vitro, but the mechanism of inhibition has not been determined. The aim of this study is to investigate the mechanism of anti-lymphangiogenesis of shikonin in primary human lymphatic endothelial cells (HMVEC-dLy). Shikonin, at non-toxic concentrations, significantly inhibited cord formation ability of lymphatic endothelial cells in a dose- and time-dependent manner. Western blotting analysis showed that shikonin decreased nuclear factor-kappaB (NF-κB) activation, as indicated by phosphorylation and nuclear translocation of NF-κB p65, and also reduced both mRNA and protein levels of hypoxia-inducible factor-1 (HIF-1)α. Use of an NF-κB inhibitor (BAY 11-7085) and HIF-1α small interfering RNA (siRNA) transfection revealed that NF-κB activation was upstream of HIF-1α expression, which controls cord formation by HMVEC-dLy. In addition, the reduction of vascular endothelial growth factor C (VEGF-C) and vascular endothelial growth factor receptor-3 (VEGFR-3) mRNA levels were also found in HMVEC-dLy that treated with shikonin. In conclusion, shikonin inhibits lymphangiogenesis in vitro by interfering the NF-κB/HIF-1α pathway and involves in suppression of VEGF-C and VEGFR-3 mRNA expression.

Oncogenic roles of serine-threonine kinase receptor-associated protein (STRAP) in osteosarcoma.

PURPOSE: To validate the presence of serine-threonine kinase receptor-associated Protein (STRAP) in osteosarcoma tissue and to investigate the oncological role of STRAP in osteosarcoma. METHODS: Expression of STRAP protein in osteosarcoma tissue compared to soft callus (hyperactive bone healing tissue) and in multiple cell lines was examined using western blot analysis. Effects of STRAP silencing on cell proliferation, invasion, migration and re-implantability in chick chorioallantoic membrane (CAM) were observed in osteosarcoma cell lines (MNNG-HOS, 143B, and U2OS). RESULTS: The result demonstrated that STRAP was highly up-regulated in osteosarcoma tissues compared with the normal physiological bone healing tissue (soft callus). Expression level of STRAP was markedly high in osteosarcoma cell lines with aggressive phenotype. Upon STRAP silencing, invasion and migration, but not proliferative activity, were selectively modulated in high-expression-STRAP cell lines. In addition, STRAP silencing reduced the success rate of tumor implantation and growth of MNNG-HOS cells in CAM model. CONCLUSIONS: Serine-threonine kinase receptor-associated protein is up-regulated during osteosarcoma progression. The presence of STRAP enhances osteosarcoma cell invasion, migration and re-implantation ability, factors which play a critical role in metastasis. Serine-threonine kinase receptor-associated protein and its related pathway are worthy for further exploration as a novel target for anti-metastasis agents.

Tumor Susceptibility Gene 101 Mediates Anoikis Resistance of Metastatic Thyroid Cancer Cells.

BACKGROUND/AIM: Resistance to anoikis is a pre-requisite step in metastasis, a major cause of death in patients with cancer, including thyroid cancer. Impairing anoikis resistance is a possible strategy for therapy of metastatic cancer. We, therefore, we aimed to investigate the key players of anoikis resistance. MATERIALS AND METHODS: Papillary-type (BCPAP), follicular-type (FTC133), and anaplastic-type (ARO) thyroid carcinoma cells, cultured in poly(2-hydroxyethyl methacrylate)-coated plates to mimic circulating cells, were used as model systems in this study. Flow cytometry and soft-agar assays were used to determine cells exhibiting anoikis resistance. Proteomics was used to identify candidate proteins and validated using western blot and siRNA knockdown. RESULTS: Only ARO cells showed both anoikis resistance potential and anchorage-independent growth ability. Tumor susceptibility gene 101 protein (TSG101) was identified to be potentially important in anoikis resistance, which was confirmed by an increase in anoikis and expression of a pro-apoptotic protein (BCL-2 like protein 4) and an apoptotic marker (cleaved poly-ADP ribose polymerase) in floating siTSG101-knockdown cells. CONCLUSION: To our knowledge, this is the first study that implicates the importance of TSG101 in anoikis resistance of thyroid cancer.

Decreasing O-GlcNAcylation affects the malignant transformation of MCF-7 cells via Hsp27 expression and its O-GlcNAc modification.

O-GlcNAcylation is a dynamic posttranslational modification of nucleoplasmic proteins. Previously, we reported that the O-GlcNAcylation level was increased in primary breast and colorectal cancer tissues. However, its precise roles in cancer development and progression are still largely unexplored. The aim of the present study was to investigate the roles of O-GlcNAcylation in the malignant transformation of cancer cell lines. O-GlcNAcylation level was examined in six cancer cell lines including breast (MCF-7 and MDA-MB-231), colorectal (SW480 and SW620), and liver (SK-Hep1 and HepG2). We found that the levels of O-GlcNAcylation and O-GlcNAc transferase (OGT), an O-GlcNAc catalyzing enzyme, were obviously increased in all cancerous cells, except SK-Hep1, when compared to normal cells. Reducing O-GlcNAcylation using RNA interference against OGT showed a marked reduction in OGT and O-GlcNAcylation levels. Surprisingly, siOGT had no effect on cell growth under conventional monolayer cultures. However, it inhibited anchorage-independent growth in soft agar cultures of all cancer cells, except SK-Hep1. Under anoikis resistance conditions performed by spheroid cultures, siOGT treatment decreased viability only in MCF-7, SW480, and SW620 cells. Among them, OGT knockdown in MCF-7 cells revealed a high inhibitory effect on colony and spheroid cultures. Using two-dimensional gel electrophoresis and mass spectrometric analysis, heat shock protein 27 (Hsp27) was found to be the highest upregulated protein upon OGT knockdown. Immunoblots revealed that the Hsp27 protein level was increased but its O-GlcNAc modification level was decreased in siOGT-treated cells. These changes were associated with the inhibition of MCF-7 cell transformation. Notably, double knockdown of OGT and Hsp27 showed a reversal in the inhibitory effect on colony and spheroid cultures. Collectively, these results indicate that O-GlcNAcylation is required for anoikis resistance and anchorage-independent growth of MCF-7 cells. Blocking this glycosylation by OGT knockdown may regulate both Hsp27 protein expression and its O-GlcNAc modification levels. This alteration may play vital roles in malignant transformation.