Kaempferol Inhibits Cervical Cancer Cells by Inducing Apoptosis and Autophagy via Inactivation of the PI3K/AKT/mTOR Signaling Pathway.

BACKGROUND/AIM: Kaempferol, a natural flavonoid, occurs abundantly in fruits and vegetables. It has various bioactivities, with antioxidant, anti-inflammatory, and other beneficial properties. The aim of this study was to investigate the in vitro effects of kaempferol on the proliferation, apoptosis, and autophagy of KB cells, a human cervical cancer cell line, and the corresponding action mechanisms. MATERIALS AND METHODS: The inhibitory efficacy of kaempferol on KB cervical cancer cells was investigated through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, migration assay, 4',6-diamidino-2-phenylindole staining, flow cytometry, acridine orange staining and western blotting. RESULTS: Kaempferol reduced KB cell viability and migration in a dose-dependent manner. Additionally, kaempferol-induced apoptosis was confirmed, and kaempferol treatment influenced levels of apoptotic proteins. Autophagy was detected upon visualization of characteristic autophagic vacuoles and acidic vesicular organelles, and verified using western blotting, which revealed elevated levels of autophagy-related proteins. Kaempferol-mediated apoptosis and autophagy were evidently attributable to reduced phosphorylation in the phosphoinositide 3-kinase (PI3K)/serine/threonine kinase 1 (AKT)/mammalian target of rapamycin (mTOR) pathway. This finding was validated using a pharmacological inhibition assay with the PI3K pathway inhibitor LY294002, which promoted KB cell apoptosis and autophagy. CONCLUSION: Our results suggest that kaempferol induces apoptosis and autophagy by inhibiting the PI3K/AKT/mTOR pathway in human cervical cancer cells, empirically showing the anticancer effects of kaempferol, and thereby presenting it as a potential anticancer therapeutic agent.

Hesperidin Inhibits Oral Cancer Cell Growth via Apoptosis and Inflammatory Signaling-Mediated Mechanisms: Evidence From In Vitro and In Silico Analyses.

Background Oral carcinoma presents a significant health challenge, prompting the need for innovative therapeutic approaches. Elevation of inflammatory mediators, including tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), has promoted cellular proliferation, inhibited apoptosis, and fostered oral cancer progression through complex signaling pathways. Hesperidin, a flavanone glycoside found in citrus fruits, is of keen interest in this study as it has been proven to have multiple health benefits through in vivo and in vitro studies. However, the mechanism behind the anticancer activity of hesperidin in oral carcinoma remains obscure. Aim The study aimed to explore the anticancer potential of hesperidin on human oral cancer cells (KB cells) by modulating pro-inflammatory and apoptotic signaling mechanisms. Methods Cancer cell growth inhibitory activity was assessed using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay. Gene expression analysis was performed using real-time RT-PCR analysis. In addition, in silico docking analysis was conducted to confirm the binding affinity of hesperidin with pro-inflammatory and apoptosis signaling molecules. The data were analyzed using one-way ANOVA and the "t" test. Results Utilizing the MTT assay, a dose-dependent cytotoxic effect of hesperidin was unveiled, with a remarkable IC50 value indicative of its potent inhibition of cell proliferation. Complementing these findings (p<0.05), qRT-PCR analysis demonstrated hesperidin's regulatory influence on key molecular targets within the KB cell line. Hesperidin treatment resulted in a noteworthy reduction in TNF-α, interleukin-1 beta (IL-1-ß), IL-6, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and B-cell lymphoma 2 (Bcl-2) mRNA expression levels (p<0.05), highlighting its inhibitory role in cell proliferation, migration, and inflammation processes. Simultaneously, hesperidin promoted the expression of BAX mRNA (p<0.05), indicating an enhancement in cell death. Molecular docking simulations further revealed robust binding affinities between hesperidin and target proteins, suggesting its potential to disrupt cellular functions and inflammatory signaling pathways in oral cancer cells. Conclusion The cytotoxic effects on the KB cell line and its anti-inflammatory properties position hesperidin as a compelling candidate for further exploration in the quest for effective oral carcinoma treatments. These findings shed light on the intricate molecular mechanisms underlying hesperidin's promise as a therapeutic agent against oral carcinoma.

Unlocking the potential of beta sitosterol: Augmenting the suppression of oral cancer cells through extrinsic and intrinsic signalling mechanisms.

The global increase in the prevalence of oral neoplasms and related deaths can be attributed to social development and lifestyle factors, leading to poor prognosis and a lack of early clinical detection. Oral cancer ranks ranked sixth mostly diagnosed cancer and is a leading cause of cancer-related deaths. In light of these circumstances, our objective was to assess the potential of ß-sitosterol, a naturally occurring herbal compound, as an anticancer agent against KB cells, a representative cell line for oral cancer. Our study primarily focused on evaluating the cytotoxic effect and mRNA expression of apoptotic proteins by ß-sitosterol on KB cells. The results demonstrated a remarkable cytotoxic effect, leading to cell death. Further investigation using flow cytometric analysis revealed that this cell death was mediated through the initiation of the apoptotic signalling by ß-sitosterol. The use of the bioinformatic tool, STITCH, supported our study by predicting drug-protein interactions and suggesting that ß-sitosterol may play a significant role in targeting apoptotic pathways. Additionally, docking results were employed to validate the findings demonstrating high binding affinity of ß-sitosterol with apoptotic-mediated signalling targets. To gain deeper insights into the molecular insights, we measured mRNA levels for BAX, BCL-2, MCL-1, P53, P21, MDM2, caspase3, and caspase9. Based on our comprehensive findings, our study concludes that ß-sitosterol holds significant therapeutic potential against oral cancer cells. These results strongly suggest that this herbal compound should be further explored as a potential treatment option for oral cancer for clinical trial.

The Combination of Photothermal Therapy and Chemotherapy using Alginate-Modified Iron Oxide-Gold Nanohybrids Carrying Cisplatin.

Background: Chemotherapy is typically the first-line treatment for the advanced stage of cancers. However, there are shortcomings with respect to conventional chemotherapy that limit therapeutic efficiency, including lack of tumor selectivity, systemic toxicity and drug resistance. Objective: A multifunctional nanoplatform was build using of hydrogel co-loaded containing cisplatin and Iron oxide-gold core-shell nanoparticles. The Au shell comprises the light response and the iron core can be utilized as a negative contrast agent in nanocomplex. Material and Methods: In this experimental study, KB cells derived from the epithelial cells located in the nasopharynx were exposed to different levels of concentration of hydrogel co-loaded with cisplatin and Iron oxide-gold core-shell nanoparticles. Afterwards, the cytotoxicity was determined using MTT assay. Results: The cytotoxicity results showed that this nanoplatforms has potent to create higher cytotoxicity in KB cells than free cisplatin, so that Fe-Au@Alg and Fe-Au@Alg with cisplatin mixed with laser irradiation exhibited a significant reduction in cell viability after 5 min. Conclusion: Hydrogel co-loaded with cisplatin and Iron oxide-gold core-shell nanoparticles are stable construct to combine chemo-photothermal therapy. Therefore, they can be used as a computed tomography-traceable nanocarrie, enabling us to monitor the delivery of therapeutics.

Cytotoxicity Analysis for the Hydroxyl Functionalized MWCNT Reinforced PMMA Nanocomposites in Oral Squamous Carcinoma (KB) Cells.

In this particular research study, a unique three-dimensional mixing technique was used to incorporate multi-walled carbon nanotubes (MWCNTs) into polymethyl methacrylate (PMMA), and the KB cell line was used in the analysis of cytotoxicity, apoptosis detection, and cell viability using the MTT assay protocol. At low concentrations (0.001 to 0.1 g/mL), these results showed that the CNT did not seem to cause cell death or apoptosis directly. It increased lymphocyte-mediated cytotoxicity against KB cell lines. This was demonstrated by the fact that the CNT increased the time it took for KB cell lines to die. In the end, the unique three-dimensional mixing method solves problems such as clumping and uneven mixing that have been written about in the relevant literature. Phagocytic uptake of MWCNT-reinforced PMMA nanocomposite by KB cells leads to oxidative stress and apoptosis induction in a dose-dependent manner. The cytotoxicity of the generated composite and the ROS (reactive oxygen species) it produces may be controlled by adjusting the MWCNT loading. The conclusion that can be drawn from the studies to date is that it could be possible to treat some types of cancer using PMMA that has MWCNTs incorporated into it.

Nanostructured lipid carrier-loaded metformin hydrochloride: Design, optimization, characterization, assessment of cytotoxicity and ROS evaluation.

Metformin hydrochloride (MET) is commonly used in diabetes treatment. Recently, it has gained interest for its anticancer potential against a wide range of cancers. Owing to its hydrophilic nature, the delivery and clinical actions of MET are limited. Therefore, the present work aims to develop MET-encapsulated NLCs using the hot-melt emulsification and probe-sonication method. The optimization was accomplished by 33 BB design wherein lipid ratio, surfactant concentration, and sonication time were independent variables while the PS (nm), PDI, and EE (%) were dependent variables. The PS, PDI, % EE and ZP of optimized GMSMET-NLCs were found to be 114.9 ± 1.32 nm, 0.268 ± 0.04 %, 60.10 ± 2.23 %, and ZP - 15.76 mV, respectively. The morphological features, DSC and PXRD, and FTIR analyses suggested the confirmation of formation of the NLCs. Besides, optimized GMSMET-NLCs showed up to 88 % MET release in 24 h. Moreover, GMSMET-NLCs showed significant cell cytotoxicity against KB oral cancer cells compared with MET solution as shown by the reduction of IC50 values. Additionally, GMSMET-NLCs displayed significantly increased intracellular ROS levels suggesting the GMSMET-NLCs induced cell death in KB cells. GMSMET-NLCs can therefore be explored to deliver MET through different routes of administration for the effective treatment of oral cancer.

Biochemical and molecular anticancer approaches for Boerhaavia diffusa root extracts in oral cancer.

Background: Boerhaavia diffusa is a medicinal herb with anti-inflammatory, antiproliferative, anticancer, and immunomodulatory properties, found across India. Aim and Objectives: The present study is designed to investigate the therapeutic potential for B. diffusa root extracts in oral cancer cell line. Materials and Methods: The aqueous and methanolic extracts of B. diffusa were prepared using Soxhlet apparatus. In order to determine the phytochemical constituents of B. diffusa, the extracts were subjected to gas chromatography-mass spectrometry analysis. The antioxidant potential of B. diffusa extracts was assessed by 2,2-Diphenyl-picrylhydrazyl, ferric ion-reducing antioxidant power, catalase and peroxidase assays. The effective concentration of B. diffusa root on cell viability was analyzed by [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The ability of B. diffusa root extracts to modify the cell-cycle phases was performed by FACS analysis. The apoptotic inducing potential of B. diffusa in oral cancer cells was confirmed by acridine orange-ethidium bromide and 4',6-diamidino-2-phenylindole staining. The protein profile of apoptotic processes was validated by the Western blot analysis; docking studies were also performed. Results: We observed that antioxidant activity was higher in B. diffusa methanolic extract compared with aqueous extract. The results showed that the methanolic and aqueous extracts of B. diffusa exhibited significant cytotoxic effect with IC50 value of 36 µg/ml and 30 µg/ml, respectively. The apoptotic DNA fragmentation and the apoptotic inducing potential in KB oral cancer cell line were higher for the methanolic extract compared with the aqueous extract. These results were also confirmed by in-silico analysis. Conclusion: The results indicate that extracts obtained from the roots of B. diffusa inhibit the progression of oral cancer. These compounds of pharmacological importance can be either used alone or in combination with other drugs to treat oral cancer.

Genetic analysis of human parechovirus type 5 isolated from children in Sapporo, Japan in the summer of 2018.

Human parechovirus (HPeV) types 1 and 3 are frequently detected in Japan, but HPeV5 is not detected. HPeV5 was isolated for the first time in Japan from seven clinical samples collected from children in Sapporo as part of the National Epidemiological Surveillance of Infectious Diseases from July to August in 2018. Seven HPeV5 strains that were detected in Sapporo (HPeV5 Sa) were analyzed in the VP1 region by direct sequencing using Sanger sequencing methods. Whole genome sequence of these strains was determined by next-generation sequencing. The VP1 region of HPeV5 Sa was closely related to HPeV5 strains detected in Belarus and Germany in 2018, and to those detected in Australia in 2019. The 3D polymerase region of HPeV5 Sa strains showed a high nucleotide identity to HPeV3 strain detected in Australia in 2013. These findings suggest that HPeV5 Sa is a recombinant virus of HPeV5 and HPeV3, and HPeV5 strains that are genetically closely related to each other may have circulated in Europe, Japan, and Australia between 2018 and 2019.

Regulation of Jun and Fos AP-1 transcription factors by JNK MAPKs signaling cascade in areca nut extract treated KB cells.

The edible endosperm of Areca catechu is recognized as a potent carcinogenic agent either consumed alone or in combination with tobacco. Habitual chewing of areca nut leads to orally potential malignant disorders which are highly effective in malignant transformation and thereby lead to oral carcinogenesis. Human buccal epithelial KB carcinoma cells were used as an experimental cell system to inspect the mechanistic act of aqueous extract of areca nut on biochemical status and their implications on transcriptional activation of cancer signaling cascade that could possibly trigger numerous oncogenic players and finally decides the cells fate. Extract treated cells showed reduced viability with altered balance between oxidants and antioxidants which lead to redox status and which is known to distort various biological processes within the cell system. Results of RT-PCR demonstrated decreased expression of BCl2, cell cycle regulators along with Activator Protein -1 (AP-1) components. While Bax, p16 and p21 mRNAs showed increased expression in extract treated KB cells. Likewise, the translational levels of proliferation cell nuclear antigen (PCNA), tumor suppressor p53, retinoblastoma (Rb) and cyclin dependent kinase 4 (CDK4) were decreased along with AP-1 subunits (c-Jun/c-Fos) with increased protein levels of p21 in extract treated KB cells. Further, the downstream activation and regulation of AP-1 transcription factors could be through stress activated c-Jun - N terminal Kinase (JNK) Mitogen Activated Protein Kinases (MAPKs) which downregulated both Jun and Fos mRNA transcripts in areca nut extract exposed KB cells. Thus, outcome of the study provides insights into mechanistic path of pathogenesis of areca related disorders. Further, it could aid in designing new therapeutic modalities that specific targets these oncogenic players and help in disease management.

Cytotoxic and larvicidal activities of Stemona alkaloids from the aerial parts and roots of Stemona curtisii Hook.f.

A new Stemona alkaloid glycoside derivative, 6-hydroxy-5,6-seco-stemocurtisinoside (4), was isolated from the ethanolic extract of the aerial parts of Stemona curtisii Hook.f., together with stemocurtisine (1), (11Z)-1',2'-didehydrostemofoline (2) and 6-hydroxy-5,6-seco-stemocurtisine (3). Whereas, stemocurtisine (1), stemocurtisinol (5) and oxyprotostemonine (6) were isolated from the roots. Their structures were elucidated using 1D and 2D NMR spectroscopy as well as MS experiments. The extract and the pure isolated compounds were evaluated for their cytotoxicities and their larvicidal activities against the dengue vector, Aedes aegypti. The alkaloid 2 showed the strongest larvicidal activity with a LC50 value of 2.44 µM. While the alkaloid 3 exhibited cytotoxicity against MCF-7 and KB cells (IC50 values of 62.52 and 18.82 µM, respectively) and showed no significant cytotoxicity against Vero cells. Additionally, quantitative analysis of the most active compounds; 2 and 3 in the crude extracts was also performed by HPLC.

Pharmacokinetic Properties of 68Ga-labelled Folic Acid Conjugates: Improvement Using HEHE Tag.

The folate receptor (FR) is a promising cell membrane-associated target for molecular imaging and radionuclide therapy of cancer (FR-α) and potentially also inflammatory diseases (FR-ß) through use of folic acid-based radioconjugate. FR is often overexpressed by cells of epithelial tumors, including tumors of ovary, cervix, endometrium, lungs, kidneys, etc. In healthy tissues, FR can be found in small numbers by the epithelial cells, mainly in the kidneys. Extremely high undesired accumulation of the folate radioconjugates in the renal tissue is a main drawback of FR-targeting concept. In the course of this work, we aimed to reduce the undesirable accumulation of folate radioconjugates in the kidneys by introducing a histidine/glutamic acid tag into their structure. Two folic acid based compounds were synthesized: NODAGA-1,4-butanediamine-folic acid (FA-I, as control) and NODAGA-[Lys-(HE)2]-folic acid (FA-II) which contains a (His-Glu)2 fragment. In vitro studies with FR (+) cells (KB and others) showed that both compounds have specificity for FR. Introduction of (HE)2-tag does not affect FR binding ability of the conjugates. In vivo biodistribution studies with normal laboratory animals, as well as with KB tumor bearing animals, were carried out. The results showed that introduction of the (HE)2 tag into the structure of folate radioconjugates can significantly reduce the accumulation of these compounds in non-target tissues and important organs (the accumulation in the kidneys is reduced 2-4 times), leaving the accumulation in tumor at least at the same level, and even increasing it.

Cordyceps militaris Fraction induces apoptosis and G2/M Arrest via c-Jun N-Terminal kinase signaling pathway in oral squamous carcinoma KB Cells.

BACKGROUND: Cordyceps militaris fraction (CMF) has been shown to possess in vitro antitumor activity against human chronic myeloid leukemia K562 cells in our previous research. MATERIALS AND METHODS: The in vitro inhibitory activities of CMF on the growth of KB cells were evaluated by viability assay. The apoptotic and cell cycle influences of CMF were detected by 4',6-diamidino-2-phenylindole staining and flow cytometry assay. The expression of different apoptosis-associated proteins and cell cycle regulatory proteins was examined by Western blot assay. The nuclear localization of c-Jun was observed by fluorescence staining. OBJECTIVE: The objective of this study was to investigate the antiproliferative effect of CMF as well as the mechanism underlying the apoptosis and cell cycle arrest it induces in KB cells. RESULTS: CMF suppressed KB cells' proliferation in a dose- and time-dependent manner. Flow cytometric analysis indicated that CMF induced G2/M cell cycle arrest and apoptosis. Western blot analysis revealed that CMF induced caspase-3, caspase-9, and PARP cleavages, and increased the Bax/Bcl-2 ratio. CMF also led to increased expression of p21, decreased expression of cyclin B1, mitotic phosphatase cdc25c, and mitotic kinase cdc2, as well as unchanged expression of p53. In addition, CMF stimulated c-Jun N-terminal kinases (JNK) protein phosphorylations, resulting in upregulated expression of c-Jun and nuclear localization of c-Jun. Pretreatment with JNK inhibitor SP600125 suppressed CMF-induced apoptosis and G2/M arrest. CONCLUSIONS: CMF is capable of modulating c-Jun caspase and Bcl-2 family proteins through JNK-dependent apoptosis, which results in G2/M phase arrest in KB cells. CMF could be developed as a promising candidate for the new antitumor agents. SUMMARY: CMF exhibited strong anticancer activity against oral squamous carcinoma KB cellsCMF inhibited KB cells' proliferation via induction of apoptosis and G2/M cell cycle arrestCMF activated JNK signaling pathway and promoted the nuclear localization of c-JunCMF regulated the apoptosis- and cell cycle-related proteins in a manner dependent on JNK/c-Jun pathway. Abbreviations used: CMF: Cordyceps militaris fraction; OSCC: Oral squamous cell carcinoma; JNK: c-Jun N-terminal kinase.

Investigating the Cytotoxicity of Folate-Conjugated Bismuth Oxide Nanoparticles on KB and A549 Cell Lines.

Purpose: Lately, bismuth-based nanomaterials have been widely utilized in medical researches such as imaging, drug delivery and radio-sensitization. Despite their advantages, bismuth-based compounds have shown toxic effects in humans. There are few studies on cytotoxicity effects of bismuth oxide (Bi2O3) nanoparticles (NPs) in-vitro. In this study, we aimed to investigate cytotoxicity of bare and also folate and 5-aminolevulinic acid (5-ALA)-conjugated Bi2O3 NPs on nasopharyngeal carcinoma (KB) and lung cancer (A549) cell lines. Methods: Bi2O3 NPs were synthesized and conjugated with folate and 5-ALA. KB and A549 cells were cultured and incubated with 10, 20, 50 and 100 µg/ml concentrations of bare and folate-5-ALA-conjugated NPs. The survival rates were obtained after 2 and 24 hours incubation of the cells with NPs using MTT assay. Also, apoptosis and ROS generation induced by the NPs in the treated cells were obtained using Caspases-3 activity assay and flow cytometry analysis, respectively. Results: Bi2O3 NPs were successfully synthesized with average size of 19.2 ± 6.5 nm, then conjugated with 5-ALA and folate. Either naked or folate-conjugated NPs were easily taken up by the cells in a concentration-dependent manner and showed cytotoxic effects. The significant cell death was noted at the concentrations more than 50 µg/ml for both compounds. Conclusion: Results indicated low cytotoxicity of the prepared NPs at lower incubation periods, which is very important for their further applications. However, 24 hours incubation of the cells with both forms of NPs caused more cell killing and the cytotoxicity increased with increasing concentrations of the NPs.

Cytotoxic Effect of Thymus caramanicus Jalas on Human Oral Epidermoid Carcinoma KB Cells

Abstract Identifying new chemotherapeutic agents with fewer side effects is a major concern for scientists today. Thymus caramanicus Jalas (Lamiaceae family) is one of the species of Thymus that grows wild in different regions of Iran. Traditionally, leaves of this plant are used in the treatment of diabetes, arthritis and cancer. Here was investigated the cytotoxic property of Thymus caramanicus essential oil and extract in human oral epidermoid carcinoma KB cells. Cell viability was measured by MTT and neutral red assays. The cells were exposed to different concentrations of essential oil (0.05-1 µL/mL) and extract (25-150 µg/mL) for 24 h. Doxorubicin was used as anticancer control drug. The data showed that the essential oil (IC50=0.44 µL/mL) and extract (IC50=105 µg/mL) induce potent cytotoxic property. Surprisingly, cytotoxic effects of essential oil and extract of this plant on KB cancer cells were greater than those on normal gingival HGF1-PI1 cell line. In addition, Thymus caramanicus could potentiate the effect of doxorubicin in sub-effective concentrations. The results of the present study indicate that essential oils and extracts of Thymus caramanicus have potential anti-proliferative property on KB cells and can be used as pharmaceutical case study for oral cancer treatments.

Resumo A identificação de novos agentes quimioterápicos com menos efeitos colaterais é uma grande preocupação para os cientistas de hoje. Thymus caramanicus Jalas (família Lamiaceae) é uma das espécies de Thymus que cresce selvagem em diferentes regiões do Irã. Tradicionalmente, as folhas desta planta são utilizados no tratamento da diabetes, artrite e câncer. Aqui investigamos a propriedade citotóxica do óleo essencial e extrato de Thymus caramanicus em células da linhagem celular tumoral humana de carcinoma epidermóide de boca (KB). A viabilidade celular foi medida por ensaios MTT e vermelho neutro. As células foram expostas a diferentes concentrações de óleo essencial (0,05-1 μL/mL) e extrato (25-150 μg/mL) durante 24 h. A doxorrubicina foi utilizada como droga de controle anticâncer. Os dados mostraram que o óleo essencial (IC50 = 0,44 μL/mL) e o extrato (IC50 = 105 μg/mL) induzem uma potente propriedade citotóxica. Surpreendentemente, os efeitos citotóxicos de óleo essencial e extrato desta planta sobre células cancerígenas KB foram maiores que sobre a linhagem celular gengival normal HGF1-PI1. Além disso, Thymus caramanicus poderia potencializar o efeito da doxorrubicina em concentrações sub-efetivas. Os resultados do presente estudo indicam que óleos essenciais e extratos de Thymus caramanicus têm potenciais propriedades anti-proliferativas sobre células KB e podem ser usado como estudos de caso farmacêuticos para tratamentos de câncer bucal

The impact of smoking on levels of chronic periodontitis-associated biomarkers.

OBJECTIVE: To investigate the effect of smoking on the expression levels of matrix metalloproteinase (MMP)-1, MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-1), and the concentrations of TNF-α and IL-10 in patients with chronic periodontitis (ChP). METHODS: This is an ex-vivo study. Our study consisted of 78 cases, all of which were diagnosed with ChP and were selected according to the inclusion and exclusion criteria. Among these 78 cases, 38 patients were classified into the smoking group (S-ChP group), and 40 patients in the non-smoking group (NS-ChP group). The clinical periodontal parameters of all patients were recorded, including the plaque index (PLI), probing depth (PD), loss of attachment (LA) and sulcus bleeding index (SBI). Serum was collected from forearm blood to establish a Porphyromonas gingivalis (Pg) internalizing KB cell model. Enzyme-linked immunosorbent assay (ELISA) was used to determine the concentrations of MMP-1, MMP-9 and TIMP-1 in the KB cell lysis solution as well as IL-10 and TNF-α in the gingival crevicular fluid (GCF). RESULTS: Fewer Pg internalizing KB cell colonies were observed in the NS-ChP group than in the S-ChP group (P<0.01). When 400µL serum was added, there were remarkable differences in the concentrations of MMP-1 and TIMP-1 secreted from the KB cells between the S-ChP and NS-ChP groups (MMP-1: t=-21.71, P<0.01; TIMP-1: t=64.35, P<0.001). Additionally, when 800µL serum was added, there were significant differences in the concentrations of MMP-1, MMP-9 and TIMP-1 in the KB cells between the S-ChP and NS-ChP groups (MMP-1: t=-81.89, P<0.001; MMP-9: t=-15.67, P<0.001; TIMP-1: t=109.4, P<0.001). The TNF-α levels were higher, but the IL-10 levels were lower in the GCF from the ChP patients in the S-ChP group than those in the NS-ChP group (both P<0.001). CONCLUSION: The serum of S-ChP patients can enhance the concentrations of MMP-1 and MMP-9, but reduce TIMP-1 secreted from Pg internalizing KB cells. However, the concentration of TNF-α was increased and IL-10 was decreased. Abnormal concentrations of ChP-associated biomarkers may be conducive to the development and progression of ChP.

Synthesis, structures, molecular docking, cytotoxicity and bioimaging studies of two novel Zn(II) complexes.

Two novel compounds [Zn2(Endc)2(bipy)2(H2O)3]·4(H2O)·2(O)(1), [Zn2(Endc)2(phen)2(H2O)]·(O)(2) (bipy = 2,2-bipyridine, phen = 1,10-phenanthroline, and Endc = endo-norbornene-cis-5,6-dicarboxylicacid) have been synthesized and characterized. In this paper abbreviations are FS-DNA (fish sperm DNA), HeLa (human cervix epithelia carcinoma cells), KB (human oral epithelial carcinoma cells), LO2 (human liver cell L-O2), EtBr (ethidium bromide), DMF (Dimethyl Formamide), MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium]). The binding of complexes with Fish Sperm DNA were measured by electronic absorption spectra and fluorescence spectroscopy. The ability of these complexes to cleave the pBR322 plasmid DNA or the KB and HeLa DNA extracted in our laboratory was demonstrated by gel electrophoresis assay. The cytotoxic effects of these complexes were examined on two tumor cell lines, HeLa, KBr and one normal cell line LO-2. UV absorption and fluorescence spectra indicate the ability of the complexes bond to DNA with different binding affinity. Gel electrophoresis assay demonstrates which one complex more effective DNA-cleavage activity. The cytotoxic activity of the complexes was tested against two different cancer and one normal cell lines. The two complexes exhibited cytotoxic specificity and significant cancer cell inhibitory rate and lower cytotoxicity toward the normal cell lines. The unique interaction mode with DNA and cancer cells inhibition effect clearly revealed the relationship between the structure and the activity of the novel antitumor agent Zn(II) complexes.

(64)Cu- and (68)Ga-Based PET Imaging of Folate Receptor-Positive Tumors: Development and Evaluation of an Albumin-Binding NODAGA-Folate.

A number of folate-based radioconjugates have been synthesized and evaluated for nuclear imaging purposes of folate receptor (FR)-positive tumors and potential therapeutic application. A common shortcoming of radiofolates is, however, a significant accumulation of radioactivity in the kidneys. This situation has been faced by modifying the folate conjugate with an albumin-binding entity to increase the circulation time of the radiofolate, which led to significantly improved tumor-to-kidney ratios. The aim of this study was to develop an albumin-binding folate conjugate with a NODAGA-chelator (rf42) for labeling with (64)Cu and (68)Ga, allowing application for PET imaging. The folate conjugate rf42 was synthesized in 8 steps, with an overall yield of 5%. Radiolabeling with (64)Cu and (68)Ga was carried out at room temperature within 10 min resulting in (64)Cu-rf42 and (68)Ga-rf42 with >95% radiochemical purity. (64)Cu-rf42 and (68)Ga-rf42 were stable (>95% intact) in phosphate-buffered saline over more than 4 half-lives of the corresponding radionuclide. In vitro, the plasma protein-bound fraction of (64)Cu-rf42 and (68)Ga-rf42 was determined to be >96%. Cell experiments proved FR-specific uptake of both radiofolates, as it was reduced to <1% when KB tumor cells were coincubated with excess folic acid. In vivo, high accumulation of (64)Cu-rf42 and (68)Ga-rf42 was found in KB tumors of mice (14.52 ± 0.99% IA/g and 11.92 ± 1.68% IA/g, respectively) at 4 h after injection. The tumor-to-kidney ratios were in the range of 0.43-0.55 over the first 4 h of investigation. At later time points (up to 72 h p.i. of (64)Cu-rf42) the tumor-to-kidney ratio increased to 0.73. High-quality PET/CT images were obtained 2 h after injection of (64)Cu-rf42 and (68)Ga-rf42, respectively, allowing distinct visualization of tumors and kidneys. Comparison of PET/CT images obtained with (64)Cu-rf42 and a (64)Cu-labeled DOTA-folate conjugate (cm10) clearly proved the superiority of NODAGA for stable coordination of (64)Cu. (64)Cu-cm10 showed high liver uptake, most probably as a consequence of released (64)Cu(2+). The data reported in this study clearly proved the promising features of (64)Cu-rf42, particularly in terms of favorable tumor-to-kidney ratios. The relatively long half-life of (64)Cu (T1/2 = 12.7 h) matches well with the enhanced circulation time of the albumin-binding NODAGA-folate, allowing PET imaging at longer time points after injection than is possible when using (68)Ga (T1/2 = 68 min).

Aloin Inhibits Interleukin (IL)-1ß-Stimulated IL-8 Production in KB Cells.

BACKGROUND: Interleukin (IL)-1ß, which is elevated in oral diseases including gingivitis, stimulates epithelial cells to produce IL-8 and perpetuate inflammatory responses. This study investigates stimulatory effects of salivary IL-1ß in IL-8 production and determines if aloin inhibits IL-1ß-stimulated IL-8 production in epithelial cells. METHODS: Saliva was collected from volunteers to determine IL-1ß and IL-8 levels. Samples from volunteers were divided into two groups: those with low and those with high IL-1ß levels. KB cells were stimulated with IL-1ß or saliva with or without IL-1 receptor agonist or specific mitogen-activated protein kinase (MAPK) inhibitors. IL-8 production was measured by enzyme-linked immunosorbent assay (ELISA). MAPK protein expression involved in IL-1ß-induced IL-8 secretion was detected by Western blot. KB cells were pretreated with aloin, and its effect on IL-1ß-induced IL-8 production was examined by ELISA and Western blot analysis. RESULTS: Saliva with high IL-1ß strongly stimulated IL-8 production in KB cells, and IL-1 receptor agonist significantly inhibited IL-8 production. Low IL-1ß-containing saliva did not increase IL-8 production. IL-1ß treatment of KB cells induced activation of MAPK signaling molecules as well as nuclear factor-kappa B. IL-1ß-induced IL-8 production was decreased by p38 and extracellular signal-regulated kinase (ERK) inhibitor treatment. Aloin pretreatment inhibited IL-1ß-induced IL-8 production in a dose-dependent manner and inhibited activation of the p38 and ERK signaling pathway. Finally, aloin pretreatment also inhibited saliva-induced IL-8 production. CONCLUSIONS: Results indicated that IL-1ß in saliva stimulates epithelial cells to produce IL-8 and that aloin effectively inhibits salivary IL-1ß-induced IL-8 production by mitigating the p38 and ERK pathway. Therefore, aloin may be a good candidate for modulating oral inflammatory diseases.

Effect of Eudragit on In Vitro Transfection Efficiency of PEI-DNA Complexes.

AIM: Eudragit® E 100 (EE100) was used to improve the transfection efficiency of polyethylenimine (PEI). MATERIALS AND METHODS: Mobility of PEI-DNA complexes with and without EE100 were visualized by agarose gel electrophoresis and their transfection efficiencies were investigated in KB human oral carcinoma cells by flow cytometry. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to determine the viability of transfected cells. RESULTS: Gel electrophoresis illustrated formation of complete complexes at N/P ratios above 5. PEI had the highest transfection efficiency at an N/P ratio of 15, whereas in combination with EE100, the transfection efficiency was highest at an N/P ratio of 7.5. High concentrations of EE100 in combination with PEI were found to reduce cell viability. CONCLUSION: The results show a synergistic action of EE100 in transfection of DNA at low N/P ratios compared to PEI alone.

Targeted images of KB cells using folate-conjugated gold nanoparticles.

Mercaptosuccinic acid-coated gold (GM) nanoparticles were prepared and characterized by transmission electron microscopy and dynamic light scattering. Folic acid (F) was then conjugated to the GM to preferentially target oral squamous cancer (KB) cells with folate receptors expressed on their membranes and facilitate the transit of the nanoparticles across the cell membrane. Finally, a fluorescence dye (Atto) was conjugated to the nanoparticles to visualize their internalization into KB cells. After culture of the cells in a medium containing GM and folate-conjugated GM (GF), the interaction of surface-modified gold nanoparticles with KB cells was studied.