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The degradation products of crosslinked silk fibroin scaffolds induce distinct macrophage polarization and responses, without causing cytotoxicity or genotoxicity.

Dentine sensitivity is a dental problem common in individuals aged between 20 and 50 years. The most effective treatment method involves occluding the exposed dental tubules. This study focused on the synthesis of calcium phosphate nanoparticles in the form of gel to use as a proof of concept for home-treatment of sensitive teeth. In this study, calcium phosphate nanoparticles were prepared using emulsion method, in which oleic fatty acid was employed as an external phase, and sodium dodecyl sulphate (SDS) was used as a surfactant to form water-in-oil nanodroplets. Finally, in order to facilitate gel formation, the gelatin solution was introduced at the final step. The amount of gelatin varied from 5 to 15 percent by weight, which was found to have an effect on the gels’ properties and the size of calcium phosphate nanoparticles embedded in gel. Based on the characterization, the calcium phosphate nanoparticles were spherical in shape, though the size decreased as the amount of gelatin increased. The gel embedding smallest nanoparticle, that is, gel-15%G, was successfully proven to be non-toxic and able to fully occlude the dentine tubules only after overnight application. According to acid challenge, the occluded materials can resist to acid solution via redissolvation and reprecipitaion process.

Colorectal cancer is one of the leading causes of cancer-related death in Thailand and many other countries. The standard practice for curing this cancer is surgery with an adjuvant chemotherapy treatment. However, the unfavorable side effects of chemotherapeutic drugs are undeniable. Recently, protein hydrolysates and anticancer peptides have become popular alternative options for colon cancer treatment. Therefore, we aimed to screen and select the anticancer peptide candidates from the in silico pepsin hydrolysate of a Cordyceps militaris (CM) proteome using machine-learning-based prediction servers for anticancer prediction, i.e., AntiCP, iACP, and MLACP. The selected CM-anticancer peptide candidates could be an alternative treatment or co-treatment agent for colorectal cancer, reducing the use of chemotherapeutic drugs. To ensure the anticancer properties, an in vitro assay was performed with “CM-biomimetic peptides” on the non-metastatic colon cancer cell line (HT-29). According to the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay results from peptide candidate treatments at 0–400 µM, the IC50 doses of the CM-biomimetic peptide with no toxic and cancer-cell-penetrating ability, original C. militaris biomimetic peptide (C-ori), against the HT-29 cell line were 114.9 µM at 72 hours. The effects of C-ori compared to the doxorubicin, a conventional chemotherapeutic drug for colon cancer treatment, and the combination effects of both the CM-anticancer peptide and doxorubicin were observed. The results showed that C-ori increased the overall efficiency in the combination treatment with doxorubicin. According to the acridine orange/propidium iodine (AO/PI) staining assay, C-ori can induce apoptosis in HT-29 cells significantly, confirmed by chromatin condensation, membrane blebbing, apoptotic bodies, and late apoptosis which were observed under a fluorescence microscope.

Riceberry rice bran is the part of rice that has been scrubbed out during coloring process. There are various health benefits with high protein content and antioxidant ability. The hydrolyzed rice bran consists of diverse peptides that provide various bioactive properties. This work aimed to study the effect of hydrolyzed riceberry rice bran extracted on colon cancer cell lines (HT-29 and SW-620) compared to normal cell (PCS-291-010). The MTT assay result showed that our extract has less cytotoxicity on normal cell (PCS-291-010, IC50 = 6,680.00 μg/ml) compared to the colon cancer cell lines and has more effect on metastatic cancer cell line (SW-620, IC50 = 5,492.31 μg /ml) than non-metastatic cancer cell line (HT-29, IC50 =6,040.76 μg/ml). According to the DNA fragmentation pattern analysis, the ladder pattern indicated that the rice bran extract can induce the apoptosis process in SW-620 cell line. Confirmed the pattern of apoptotic cell by AO/PI double stain test and quantified apoptotic cells by Annexin V. For the cell senescence analysis, SA-β-gal staining technique was performed at 24 h after treatments, HT-29 reached maximum senescence rate at 85.74% while SW-620 had only 17.23% of senescence. And a result of cell cycle analysis, HT-29 were decreased the number of cells in S, M/G2 phase, and increased the number of cells in G0/G1 phase. Furthermore > 50 kDa peptide fraction separated from HRBE has a potent anti-cancer cells (SW-620, IC50 = 4,908 μg/ml). In conclusion, the hydrolyzed riceberry rice bran extract can inhibit colon cancer cell lines with less effect on normal cell. The extracts could induce apoptosis process in metastatic cancer cell and induce senescence process in non-metastatic cancer cell. This observed information will be useful and applicable for medical research and colon cancer treatment in the future.

Forkhead box M1 (FOXM1) is known to play a role in breast cancer progression. FOXM1 inhibition becomes one of the strategies in developing the novel cancer therapy. Recently, thiostrepton has been recognized as a potent FOXM1 inhibitor. To improve its potential, we aimed to develop a nanodelivery system for thiostrepton. Here, liposome-encapsulated thiostrepton (TSLP) was developed. Physiochemical properties were characterized by TEM and dynamic light scattering technique. The biological activities were also evaluated, by cellular internalization, MTT assay, spheroid formation assay and RT-PCR. The result showed that the range sizes of TSLP were 152 ± 2 nm, polydispersity index (PdI) of 0.23 ± 0.02 and zeta potential of −20.2 ± 0.1 mV. As expected, TSLP showed a higher potential in reducing FOXM1 levels in MCF-7 cells than free thiostrepton. Additionally, TSLP significantly improved the efficiently and specificity of thiostrepton in reducing cell viability of MCF-7, but not of the fibroblast (HDFn) cells. Interestingly, TSLP had an ability to induce MCF-7 cell death in both 2D monolayer and 3D spheroid culture. In conclusions, TSLP could possibly be one of the potential developments using nano-delivery system to improve abilities and specificity of thiostrepton in breast cancer cell inhibition and death inducing, with decreasing non-specific toxicity.

Breast cancer is the most common type of malignancies in women worldwide, and genotoxic chemotherapeutic drugs are effective by causing DNA damage in cancer cells. However, >90% of patients with metastatic cancer are resistant to chemotherapy. The Forkhead box M1 (FOXM1) transcription factor plays a pivotal role in the resistance of breast cancer cells to chemotherapy by promoting DNA damage repair following genotoxic drug treatment. The aim of the present study was to investigate the inhibition of the FOXM1 protein by thiostrepton, a natural antibiotic produced by the Streptomyces species. Experimental studies were designed to examine the effectiveness of thiostrepton in downregulating FOXM1 mRNA expression and activity, leading to senescence and apoptosis of breast cancer cells. The cytotoxicity of thiostrepton in breast cancer was determined using cell viability assay. Additionally, thiostrepton treatment decreased the mRNA expression of cyclin B1 (CCNB1), a downstream target of FOXM1. The present results indicated that thiostrepton inhibited FOXM1 mRNA expression and its effect on CCNB1. Molecular dynamic simulations were performed to study the interactions between FOXM1-DNA and thiostrepton after molecular docking. The results revealed that the possible mechanism underlying the inhibitory effect of thiostrepton on FOXM1 function was by forming a tight complex with the DNA and FOXM1 via its binding domain. Collectively, these results indicated that thiostrepton is a specific and direct inhibitor of the FOXM1 protein in breast cancer. The findings of the present study may lead to the development of novel therapeutic strategies for breast cancer and help overcome resistance to conventional chemotherapeutic drugs.

Objectives Calcium phosphate/SLS/P123 composite bioactive gels were prepared to achieve dentine tubule occlusion. Methods Gels containing calcium phosphate particles were prepared in a water-in-oil microemulsion system with a mixture of triblock copolymer pluronic (P123) as a co-surfactant and sodium lauryl sulfate (SLS) as a surfactant in cyclohexane. Subsequently, calcium chloride dihydrate and sodium hydrogen phosphate aqueous solutions were added in a water phase. Finally, slow evaporation of the oil phase at room temperature was performed to produce a hybrid gel. The obtained gels were investigated for their toxicity by the sulforhodamine B (SRB) assay and applied on human dentine specimens to examine their ability to occlude dentine tubules. Results The size and morphology of the calcium phosphate particles embedded in the gel depended on the concentration of P123 and SLS, which were used as a template for mineral precipitation. The prepared calcium phosphate particles (200–500 nm in diameter) with the maximum polymer and surfactant content exhibited spherical shapes. Further, on reducing their content twice and tenfold yields micro-particles with flower-like shapes. These bioactive gels were able to occlude into dentine tubules after 3 days of application with a plugging rate of 79.22% when using the smallest particles. In addition, calcium phosphate nanorods were transformed into dentine tubules with a maximum depth of 6 μm on increasing the amount of gel. Conclusions The bioactive gels were effectively used as bioactive fillers to occlude exposed human dentine tubules.

Essential oils (EOs) from various aromatic herbs exhibit potent in vitro antimicrobial characteristics against several bacterial foodborne pathogens and have therefore been proposed as natural alternatives to antibiotic agents. However, oral administration of EOs has limited practicality because of their high volatility, odor, rapid decomposition, and poor bioavailability in the lower part of the intestines. The main objective of the present study was to develop and evaluate an innovative tool for targeted delivery and sustained release of EOs in the gastrointestinal tract by using a nano/microstructured hybrid composite system. Cinnamon oil was used as a plant-derived antimicrobial agent model to prepare the nanoparticles via the hot homogenization technique. The prepared nanoparticles were then encapsulated in microparticles using an alginate-calcium chloride system. Their stability was investigated in simulated gastrointestinal fluid. The particles were also physicochemically characterized and biologically evaluated as antibacterial agents against foodborne pathogens. Our results showed that under simulated gastrointestinal conditions, the nano/microstructured hybrid composite particles exhibited good gastric resistance and sustained release in intestinal fluids. They also exhibited excellent antibacterial characteristics against important foodborne pathogens, namely, Escherichia coli O157:H7, Pseudomonas aeruginosa, Salmonella typhi, S. enterica, Staphylococcus aureus, and Vibrio cholerae. In conclusion, the innovation reported in the present study is an improved formula of cinnamon oil that could serve as a promising candidate as an antibacterial bioproduct for decontamination of foodborne pathogens in the gastrointestinal tract.

A cascade reaction of 3-hydroxy-2-methylpyridine 1-oxide and substituted 2-fluorobenzal-dehydes involving nucleophilic aromatic substitution (SNAr) and Knoevenagel condensation has been developed. This reaction provided easy access to benzo[6,7]oxepino[3,2-b]pyridine 1-oxide derivatives in moderate to good yields. Subsequent reduction of these compounds provided their deoxygenated forms in good to excellent yields. Moreover, some of the synthesized compounds were found to be active against human colorectal cancer cells (HCT-116 cell lines) with IC50 values in the range of 24.95–45.80 μM.

The forkhead transcription factor FOXM1 has a key role in DNA damage response, and its deregulated overexpression is associated with genotoxic drug resistance in breast cancer. However, little is known about the posttranslational mechanisms by which FOXM1 expression is regulated by genotoxic agents and how they are deregulated in resistant cells. Initial co-immunoprecipitation studies verified previous proteomic analysis finding that the OTUB1 is a novel FOXM1-interacting protein. Western blot analysis showed that both OTUB1 and FOXM1 expression reduced upon genotoxic agent treatment in MCF-7 cells, but remained relatively constant in resistant cells. FOXM1 expression reduced upon OTUB1 depletion by siRNA and increased with OTUB1 overexpression in MCF-7 cells, arguing that OTUB1 positively regulates FOXM1 expression. In agreement, co-immunoprecipitation experiments demonstrated that FOXM1 expression is associated with OTUB1 binding but inversely correlates with conjugation to the protein degradation-associated Lys-48-linked ubiquitin-chains. Overexpression of wild-type (WT) OTUB1, but not the OTUB1(C91S) mutant, disrupted the formation of Lys48-linked ubiquitin-conjugates on FOXM1. Importantly, knockdown of OTUB1 by siRNA resulted in an increase in turnover of FOXM1 in MCF-7 cells treated with the protein synthesis inhibitor cycloheximide, whereas overexpression of WT OTUB1, but not the OTUB1(C91S) mutant, significantly enhances the half-life of FOXM1. In addition, proliferative and clonogenic assays also show that OTUB1 can enhance the proliferative rate and epirubicin resistance through targeting FOXM1, as OTUB1 has little effect on FOXM1-deficient cells. The physiological relevance of the regulation of FOXM1 by OTUB1 is further underscored by the significant correlations between FOXM1 and OTUB1 expression in breast cancer patient samples. Cox-regression survival analysis indicates that OTUB1 overexpression is linked to poorer outcome in particular in patients treated with chemotherapy. Collectively, these data suggest that OTUB1 limits the ubiquitination and degradation of FOXM1 in breast cancer and has a key role in genotoxic agent resistance.

Fluorescence lifetime measurements can provide quantitative assays of the local fluorophore environment and can be applied to read out biomolecular interactions via Forster resonance energy transfer (FRET). Fluorescence lifetime imaging (FLIM) can be automated for high content analysis (HCA) to map protein–protein interactions with applications in drug discovery, systems biology and basic research. The automated acquisition of FLIM data over 100's of fields of view provides statistical power to overcome noise in instrumentation and biological systems and thus exploit relatively small changes in mean lifetime to provide useful readouts that would not be practically achievable in manual microscopy experiments. We present here an automated HCA system with the ability to perform rapid unsupervised optically sectioned FLIM of fixed and live biological samples and illustrate its potential through exemplar applications of different FRET readouts.

The novel biodegradable films of chloroacetated natural rubber/polyvinyl alcohol (CNR/PVA) (55/45 wt%) non-woven nanofiber films encapsulated with kaolin and starch (2.5 and 5 wt%) were produced successfully by green electrospinning technique. The effect of fillers with different content on the physical, chemical, mechanical, biocompatibility and biodegradation properties of CNR/PVA nanofiber films were investigated. The higher crystallinity obtained in CNR/PVA encapsulate with 2.5 wt% kaolin and nanofibers were formed with the maximum diameter distribution and mean value of 40–160 nm and 94.15 ± 54.19 nm respectively. DSC and DMA revealed the kaolin can improve the interfacial adhesion between CNR and PVA and contribute to enhancing the chemical interactions. The mechanical properties improved upon encapsulation of starch and kaolin and more favourable nanofibers with smaller diameter obtained using kaolin rather than starch. The cytotoxicity results revealed the viability of the prepared nanofiber films with human dermal fibroblast cell. Furthermore, the incorporation of starch and kaolin accelerated the degradation rate and the highest enzymatic degradation obtained with 2.5 wt% of starch. The prepared nanofiber films have the potential to be applied for the skin tissue engineering scaffold applications.

Calcium phosphate composite organogel was synthesized from a mixture of sorbitan monopalmitate as emulsifier and castor oil as oil phase at 60 °C to establish emulsion system with calcium chloride and di-sodium hydrogen phosphate aqueous solution as water phase. The heated mixture was cooled at room temperature to form organogel. The prepared organogels were investigated by thermogravimetric analysis (TGA), scanning electron microscope (SEM), transmission electron microscope (TEM) and cytotoxicity test. The prepared organogels of 4:3 and 1:1 in oil/water content ratio were white and gritty in nature, which resulted in calcium phosphate nanoparticles with spherical shape of 1.3 to 1.9 μm and 1.2 to 2.3 μm, respectively. They were able to successfully occlude into dentine tubules within 1 day. In addition, the prepared organogels were found to have acceptable toxicity level for daily human use with high concentration up to 1600 μg/ml. Hence, these organogels showed the promising results to be developed as an effective dentine occlusion material in relieving of dentin hypersensitivity.

The forkhead box transcription factor FOXM1 is an essential effector of G2/M-phase transition, mitosis and the DNA damage response. As such, it is frequently deregulated during tumorigenesis. Here we report that FOXM1 is dynamically modified by SUMO1 but not by SUMO2/3 at multiple sites. We show that FOXM1 SUMOylation is enhanced in MCF-7 breast cancer cells in response to treatment with epirubicin and mitotic inhibitors. Mutation of five consensus conjugation motifs yielded a SUMOylation-deficient mutant FOXM1. Conversely, fusion of the E2 ligase Ubc9 to FOXM1 generated an auto-SUMOylating mutant (FOXM1-Ubc9). Analysis of wild-type FOXM1 and mutants revealed that SUMOylation inhibits FOXM1 activity, promotes translocation to the cytoplasm and enhances APC/Cdh1-mediated ubiquitination and degradation. Further, expression of the SUMOylation-deficient mutant enhanced cell proliferation compared with wild-type FOXM1, whereas the FOXM1-Ubc9 fusion protein resulted in persistent cyclin B1 expression and slowed the time from mitotic entry to exit. In summary, our findings suggest that SUMOylation attenuates FOXM1 activity and causes mitotic delay in cytotoxic drug response.

FOXM1 is implicated in genotoxic drug resistance but its mechanism of action remains elusive. We show here that FOXM1-depletion can sensitize breast cancer cells and mouse embryonic fibroblasts (MEFs) into entering epirubicin-induced senescence, with the loss of long-term cell proliferation ability, the accumulation of γH2AX foci, and the induction of senescence-associated β-galactosidase activity and cell morphology. Conversely, reconstitution of FOXM1 in FOXM1-deficient MEFs alleviates the accumulation of senescence-associated γH2AX foci. We also demonstrate that FOXM1 regulates NBS1 at the transcriptional level through an forkhead response element on its promoter. Like FOXM1, NBS1 is overexpressed in the epirubicin-resistant MCF-7Epi(R) cells and its expression level is low but inducible by epirubicin in MCF-7 cells. Consistently, overexpression of FOXM1 augmented and FOXM1 depletion reduced NBS1 expression and epirubicin-induced ataxia-telangiectasia mutated (ATM)phosphorylation in breast cancer cells. Together these findings suggest that FOXM1 increases NBS1 expression and ATM phosphorylation, possibly through increasing the levels of the MRN(MRE11/RAD50/NBS1) complex. Consistent with this idea, the loss of P-ATM induction by epirubicin in the NBS1-deficient NBS1-LBI fibroblasts can be rescued by NBS1 reconstitution. Resembling FOXM1, NBS1 depletion also rendered MCF-7 and MCF-7Epi(R) cells more sensitive to epirubicin-induced cellular senescence. In agreement, the DNA repair-defective and senescence phenotypes in FOXM1-deficent cells can be effectively rescued by overexpression of NBS1. Moreover, overexpression of NBS1 and FOXM1 similarly enhanced and their depletion downregulated homologous recombination (HR) DNA repair activity. Crucially, overexpression of FOXM1 failed to augment HR activity in the background of NBS1 depletion, demonstrating that NBS1 is indispensable for the HR function of FOXM1. The physiological relevance of the regulation of NBS1 expression by FOXM1 is further underscored by the strong and significant correlation between nuclear FOXM1 and total NBS1 expression in breast cancer patient samples, further suggesting that NBS1 as a key FOXM1 target gene involved in DNA damage response, genotoxic drug resistance and DNA damage-induced senescence.

Most commonly used anticancer drugs exert their effects mainly by causing DNA damage. The enhancement in DNA damage response (DDR) is considered a key mechanism that enables cancer cells to survive through eliminating the damaged DNA lesions and thereby developing resistance to DNA-damaging agents. This chapter describes the four experimental approaches for studying DDR and genotoxic drug resistance, including the use of γ-H2AX and comet assays to monitor DNA damage and repair capacity as well as the use of clonogenic and β-galactosidase staining assays to assess long-term cell fate after DNA-damaging treatment. Finally, we also present examples of these methods currently used in our laboratory for studying the role of FOXM1 in DNA damage-induced senescence and epirubicin resistance.

Most commonly used anticancer drugs exert their effects mainly by causing DNA damage. The enhancement in DNA damage response (DDR) is considered a key mechanism that enables cancer cells to survive through eliminating the damaged DNA lesions and thereby developing resistance to DNA-damaging agents. This chapter describes the four experimental approaches for studying DDR and genotoxic drug resistance, including the use of γ-H2AX and comet assays to monitor DNA damage and repair capacity as well as the use of clonogenic and β-galactosidase staining assays to assess long-term cell fate after DNA-damaging treatment. Finally, we also present examples of these methods currently used in our laboratory for studying the role of FOXM1 in DNA damage-induced senescence and epirubicin resistance.

Human telomerase consisting of telomerase RNA template (hTR) and telomerase reverse transcriptase (hTERT) provides a mechanism for synthesis of telomere repeats that prolongs life span of cells. Telomerase activity is present in germ-line and malignant tumor cells but not in most normal human somatic cells. This study determined hTERT mRNA level in tissue samples from patients with gastrointestinal tract (GI) cancers. Tissue samples were obtained from 22 GI cancer patients, 3 gastrointestinal stomal tumors (GIST) and 25 corresponding non-cancerous tissues. hTERT expression was determined by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) using Taqman probe, hTERT mRNA was detected in 12 of 22 cancerous tissue samples. Six of 8 tissue samples obtained from patients with hepatocellular carcinoma and cholangiocarcinoma were positive for hTERT. However, hTERT mRNA was not detected in GIST and non-cancerous tissues. These results suggest that hTERT may be an effective target for cancer therapies to treat many type of GI cancers including cholangiocarcinoma and hepatocellular carcinoma.