Significant Cytostatic Effect Research Articles

Redox-responsive Nanomicelles with Intracellular Targeting and Programmable Drug Release for Targeted Tumor Therapy.

Anti-inflammatory medications, in particular aspirin, have chemopreventive and anticancer adjuvant effects on specific types of cancers, according to ongoing anti-tumor research. Additionally, efforts have been made to transform Poly(salicylic acid) (PSA) into delivery-related nanocarriers. to transport anticancer medications into nanocarriers. However, tumor cell targeting and tumor selectivity were lacking in the salicylic acid polymer-based nanocarriers, preventing them from performing to their full potential. The objective of this study is to prepare targeting and reduction-responsive poly pre-drug nanocarriers (HA-ss-PSA NPs) and to investigate the feasibility of delivering adriamycin (DOX) as nanocarriers. The structures of the polymers were confirmed by nuclear magnetic resonance hydrogen spectroscopy (1H-NMR) and infrared spectroscopy (IR); the encapsulation rate and drug loading of DOX-loaded nanoparticles were determined by HPLC; and the anti-tumor effects of the carriers were evaluated by MTT experiments and in vivo</i> experiments. The prepared nanocarriers had uniform particle size distribution. The drug release rate was up to 80% within 48 h in the tumor environment. DOX/HA-ss-PSA NPs showed significant cytostatic effects. In addition, HA-ss-PSA NPs showed significant targeting and inhibition of cell migration in cell uptake and scratch assays. In vivo</i> experiments showed that the prepared carriers had high tumor inhibition rates, good targeting effects on the liver and tumor, and significantly reduced toxicity to other tissues. The prepared HA-ss-PSA NPs could effectively inhibit the growth of HepG2 cells and tumors in vivo</i>, indicating that PSA could be used as a backbone component of a safe and reliable drug delivery system, providing a new strategy for the treatment of liver cancer.

Study of the Protective Properties of 2-Ethyl-6-Methyl-3-Hydroxypyridine Malate in the Model of In vitro-Induced Oxidative Stress in Myoblast Cell Culture

Background: A cellular model of oxidative stress induced by hydrogen peroxide in the primary culture of myoblasts was obtained by in vitro experiments, and the possibility of exogenous regulation of the cytotoxic effect using 2-ethyl-6-methyl-3-hydroxypyridine malate (ethoxidol) was studied. Moreover, the influence of oxidative stress and the effect of ethoxidol on the intracellular expression of such an important biomarker as myostatin was investigated. Methods: Hydrogen peroxide was used to induce oxidative stress. The effect of hydrogen peroxide on the rate of myoblast proliferation was studied by measuring the reduction level of (3-(4,5- dimethylthiazole-2-yl))-2,5-diphenyltetrazolium bromide. To measure the expression of myostatin, a real-time polymerase chain reaction (PCR-RV) method was used. Results: During the work, it was clearly demonstrated that hydrogen peroxide has a significant cytostatic effect on myoblasts in vitro, inhibiting their proliferation. Ethoxidol in physiological concentration did not show toxic effects and did not inhibit cell proliferation. This antioxidant revealed a statistically significant protective effect on the cytostatic effect of hydrogen peroxide on myoblasts. In addition, this compound inhibited the expression of myostatin mRNA caused by exposure to hydrogen peroxide as a negative regulator of growth and differentiation of muscle tissue that occurs in response to exposure to reactive oxygen species. Conclusion: Hydrogen peroxide is one of the highly active forms of oxygen and has a significant cytostatic effect on myoblasts in vitro, suppressing their proliferation. 2-ethyl-6-methyl-3- hydroxypyridine malate neutralizes the toxic effect of peroxide, thereby indirectly having a positive effect on the rate of myoblast proliferation in vitro.

Cytotoxic Activity of α-Aminophosphonic Derivatives Coming from the Tandem Kabachnik-Fields Reaction and Acylation.

Encouraged by the significant cytotoxic activity of simple α-aminophosphonates, a molecular library comprising phosphonoylmethyl- and phosphinoylmethyl-α-aminophosphonates, a tris derivative, and N-acylated species was established. The promising aminophosphonate derivatives were subjected to a comparative structure-activity analysis. We evaluated 12 new aminophosphonate derivatives on tumor cell cultures of different tissue origins (skin, lung, breast, and prostate). Several derivatives showed pronounced, even selective cytostatic effects. According to IC50 values, phosphinoylmethyl-aminophosphonate derivative 2e elicited a significant cytostatic effect on breast adenocarcinoma cells, but it was even more effective against prostatic carcinoma cells. Based on our data, these new compounds exhibited promising antitumor activity on different tumor types, and they might represent a new group of alternative chemotherapeutic agents.

Cytotoxic Potential of Bioactive Compounds from Aspergillus flavus, an Endophytic Fungus Isolated from Cynodon dactylon, against Breast Cancer: Experimental and Computational Approach.

Endophytic fungi are a diverse group of microorganisms that colonize the inter- or intracellular spaces of plants and exhibit mutual benefits. Their interactions with the host plant and other microbiomes are multidimensional and play a crucial role in the production of secondary metabolites. We screened bioactive compounds present in the extracts of Aspergillus flavus, an endophytic fungus isolated from the roots of the medicinal grass Cynodon dactylon, for its anticancer potential. An in vitro analysis of the Ethyl acetate extract from A. flavus showed significant cytostatic effects (IC50: 16.25 μg/mL) against breast cancer cells (MCF-7). A morphological analysis of the cells and a flow cytometry of the cells with annexin V/Propidium Iodide suggested that the extract induced apoptosis in the MCF-7 cells. The extract of A. flavus increased reactive oxygen species (ROS) generation and caused a loss of mitochondrial membrane potential in MCF-7 cells. To identify the metabolites that might be responsible for the anticancer effect, the extract was subjected to a gas chromatography-mass spectrometry (GC-MS) analysis. Interestingly, nine phytochemicals that induced cytotoxicity in the breast cancer cell line were found in the extract. The in silico molecular docking and molecular dynamics simulation studies revealed that two compounds, 2,4,7-trinitrofluorenone and 3α, 5 α-cyclo-ergosta-7,9(11), 22t-triene-6beta-ol exhibited significant binding affinities (-9.20, and -9.50 Kcal/mol, respectively) against Bcl-2, along with binding stability and intermolecular interactions of its ligand-Bcl-2 complexes. Overall, the study found that the endophytic A. flavus from C. dactylon contains plant-like bioactive compounds that have a promising effect in breast cancer.

Structural properties and evaluation of the antiproliferative activity of limonene-1,2-diol obtained by the fungal biotransformation of R-(+)- and S-(-)-limonene.

Limonene-1,2-diol is a limonene oxygenated metabolite that possesses eight different stereoisomers, which could result in different biological properties. Nonetheless, the relation between its spatial configuration and biological function is still little explored. The present study aimed to perform the stereoisomers identification using nuclear magnetic resonance (NMR) investigation of the limonene-1,2-diol produced via R-(+)- and S-(-)-limonene biotransformation by Colletotrichum nymphaeae and S-(-)-limonene biotransformation by Fusarium oxysporum 152B. Besides, in vitro antiproliferative activity was evaluated against human tumor and nontumor cell lines. The NMR analysis showed that R-(+)-limonene biotransformation afforded exclusively (+)-(1S,2S,4R-limonene-1,2-diol), whereas S-(-)-limonene biotransformation afforded exclusively (-)-(1R,2R,4S-limonene-1,2-diol) independent on the fungi used. Despite no significant cytostatic effects, a possible influence of stereogenic center on the antiproliferative activity of these limonene biotransformation products was evidenced. Moreover, the lack of in vitro antiproliferative effect of limonene-1,2-diol against nontumor cells suggested a safe dose range for further in vivo evaluations, including food applications.

Lysosome‑targeted drug combination induces multiple organelle dysfunctions and non‑canonical death in pancreatic cancer cells.

Pancreatic cancer is one of the leading causes of cancer-related mortality and has the lowest 5-year survival rate. Therefore, novel strategies are urgently required to treat pancreatic cancer. Pancreatic ductal adenocarcinoma (PDAC) cells rely on enhanced lysosomal function for survival and proliferation to facilitate the degradation of contents accumulated via autophagy and macropinocytosis. Previously, we have reported that the combination of epidermal growth factor receptor/HER2 inhibitor lapatinib and sphingosine analog fingolimod (FTY720) confers a significant cytostatic effect in lung cancer cells. In the present study, the combined effects of these drugs on PDAC cell lines, BxPC-3, KP-4, PANC-1 and MIA PaCa-2, were examined. It was observed that FTY720 enhanced the lapatinib-induced cytotoxic effect and caused non-canonical and lysosome-dependent death in PDAC cells. Lapatinib and FTY720 induced lysosomal swelling and inhibited lysosomal acidification. Combination treatment with lapatinib and FTY720 increased lysosomal membrane permeability, induced mitochondrial depolarization, induced endoplasmic reticulum stress and disturbed intracellular calcium homeostasis. Additionally, the cytotoxic effect of lapatinib was enhanced by hydroxychloroquine or the CDK4/6 inhibitor abemaciclib, both of which induce lysosomal dysfunction. Collectively, these results indicated that the lysosome-targeted drug combination induces multiple organelle dysfunction and exerts a marked cytotoxic effect in PDAC cells.

21,24-Cyclolanostanes revisited: Structural revision and biological evaluation

Chemical fractionation of the EtOH extract of a medicinal macro fungus, Inonotus obliquus, afforded an array of lanostane-type triterpenoids (1−11) including two new ones (1 and 8). The structures of these compounds were determined on the basis of spectroscopic analyses, single crystal X-ray crystallography of 3–6 and biosynthetic considerations. With the confirmatory structural information provided by X-ray diffraction analysis in hand, several previously reported 21,24-cyclolanostanes, such as inonotsutriols A–C and (20R,21S,24S)-21,24-cyclopenta-3β,21,25-trihydroxylanosta-8-ene, were structurally corrected. In addition, the NMR data of other types of 21,24-cyclo triterpenoids were also re-examined and structural revisions were thus suggested. Compounds 2, 6 and 8 showed significant cytostatic effects against a panel of tumor cell lines with IC50 values ranging from 7.80 to 18.5 μM. Further assays established that compound 2 exerted promising in vitro anti-breast cancer potential by inhibiting the proliferation and migration of 4T1 cells.

Differences in the efficiency of 3-deazathiamine and oxythiamine pyrophosphates as inhibitors of pyruvate dehydrogenase complex and growth of HeLa cells in vitro

Oxythiamine (OT) and 3-deazathiamine (DAT) are the antimetabolites of thiamine. The aim of study was to compare the effects of OT and DAT pyrophosphates (-PP) on the kinetics of mammalian pyruvate dehydrogenase complex (PDHC) and the in vitro culture of HeLa cells. The kinetic study showed that 3-deazathiamine pyrophosphate (DATPP) was a much stronger competitive inhibitor (Ki = 0.0026 μM) of PDHC than OTPP (Ki = 0.025 μM). Both Ki values were much lower versus K m for thiamine pyrophosphate (0.06 μM). However, DATPP added to the culture medium for the HeLa cells culture did not hamper the rate of cell growth and showed not significant impact on the viability of the cells, whereas OTPP and OT showed a significant cytostatic effect. The differences between the thiamine antivitamins in their effect on cell growth in vitro may be due to differences in physicochemical properties and difficulty in DAT transport across the cell membrane.

Fractionation-Dependent Radiosensitization by Molecular Targeting of Nek1.

NIMA (never-in-mitosis gene A)-related kinase 1 (Nek1) is shown to impact on different cellular pathways such as DNA repair, checkpoint activation, and apoptosis. Its role as a molecular target for radiation sensitization of malignant cells, however, remains elusive. Stably transduced doxycycline (Dox)-inducible Nek1 shRNA HeLa cervix and siRNA-transfected HCT-15 colorectal carcinoma cells were irradiated in vitro and 3D clonogenic radiation survival, residual DNA damage, cell cycle distribution, and apoptosis were analyzed. Nek1 knockdown (KD) sensitized both cell lines to ionizing radiation following a single dose irradiation and more pronounced in combination with a 6 h fractionation (3 × 2 Gy) regime. For preclinical analyses we focused on cervical cancer. Nek1 shRNA HeLa cells were grafted into NOD/SCID/IL-2Rγc−/− (NSG) mice and Nek1 KD was induced by Dox-infused drinking water resulting in a significant cytostatic effect if combined with a 6 h fractionation (3 × 2 Gy) regime. In addition, we correlated Nek1 expression in biopsies of patients with cervical cancer with histopathological parameters and clinical follow-up. Our results indicate that elevated levels of Nek1 were associated with an increased rate of local or distant failure, as well as with impaired cancer-specific and overall survival in univariate analyses and for most endpoints in multivariable analyses. Finally, findings from The Cancer Genome Atlas (TCGA) validation cohort confirmed a significant association of high Nek1 expression with a reduced disease-free survival. In conclusion, we consider Nek1 to represent a novel biomarker and potential therapeutic target for drug development in the context of optimized fractionation intervals.

Comparative Cr, As and CCA induced Cytostaticity in mice kidney: A contribution to assess CCA toxicity

CCA (Chromium Copper Arsenate) treated wood, widely used in outdoor residential structures and playgrounds, poses considerable dangers of leaching of its components to the environment.In this study, mouse kidney samples were used to evaluate the effects of CCA, chromium trioxide (CrO3) and arsenic pentoxide (As2O5) on cell pathophysiology by flow cytometry. Samples were collected after 14, 24, 48 and 96 h of animal exposure. While Cr had no statistically significant cytostatic effects, As2O5 induced a S-phase delay in animals exposed for 24 h, and over time a G0/G1 phase blockage. The effects of CCA in S-phase were similar, but more severe than those of As2O5. Since environmental and public health hazards due to the long durability of CCA-treated wood products, these data confirm that CCA has profoundly toxic effects on cell cycle, distinct from the compounds themselves. These cytostatic effects support cell cycle dynamics as a valuable endpoint to assess the toxicity of remaining CCA-treated infrastructures, and the expected increased waste stream over the coming decades.

Anticancer and biological properties of a Zn-2,6-diacetylpyridine bis(thiosemicarbazone) complex

Herein, to develop a multi-target anticancer metal agent and achieve a "1 + 1 > 2" pharmaceutical effect, we rationally designed and synthesized five complexes (C1-C5) by synergistically exploiting the properties of Zn(ii) and a series of modified 2,6-diacetylpyridine bis(thiosemicarbazone) ligands. By investigating the structure-activity relationships, we found that the binuclear Zn(ii) complex (C5) acts against human bladder cancer cells (T-24) with significant cytotoxicity. We subsequently determined the multiple anticancer mechanisms of C5 to T-24 cells, including inhibiting the activity of topoisomerase I (Topo I), blocking the cell cycle in the S phase, and inducing apoptosis and autophagy in T-24 cells. Furthermore, C5 inhibited the migration of T-24 cells and showed a significant cytostatic effect in the T-24 3D spheroid model.

Chemical Analysis and Cytotoxic and Cytostatic Effects of Twelve Honey Samples Collected from Different Regions in Morocco and Palestine.

The aim of this in vitro study is to characterize the phenolic compounds of twelve honey samples collected from different locations in Palestine (H1-6) and Morocco (H7-12) and to evaluate their cytotoxic and cytostatic effects in cells from the human colorectal carcinoma cell line HCT-116 and breast cancer cell line MCF-7. Quantitative HPLC analysis revealed nine phenolic compounds in three Moroccan honey samples, namely, syringic acid, tannic acid, caffeic acid, ferulic acid, coumaric acid, gallic acid, rosmarinic acid, epicatechin, and pyrogallol. Syringic acid, abundant in numerous types of honey with strong antioxidant capacities, was present at values ranging between 0.10 mg/100 g and 1.24 mg/100 g of Daghmos (H11) and Kabbar (H10) samples, respectively. No significant reductions in cell viability were observed in both cell lines treated with the Palestinian samples as measured with MTT assay. Significant cytostatic effects were after treatment of HCT cells with Morar honey H1 with IC50 of 1789 μg/ml. Three Moroccan samples, H7 (Zaâtar), H9 (Bochnikha), and H10 (Kabbar), showed slight, but significant cytostatic effects in HCT cells. A strong correlation was observed between cytostatic activity of MCF cells and antioxidant content (phenols, flavonoids, and flavonol). Furthermore, a strong negative correlation was detected between the cytostatic activity in HCT cells and the contents of syringic acid (r= -0.756) and tannic acid (r= -0.610). These results indicate that the traditionally known anticancer effects of honey might be mediated in part through cytostatic effects.

Response of breast cancer cells to IFNα-2b in 2D and 3D cell cultures.

The effect of IFNα-2b on the migration, proliferation, and expression of epithelial and mesenchymal markers of MCF-7 tumor adenocarcinoma cells in 2D and 3D cell cultures was examined. A significant cytostatic effect of IFNα-2b on the tumor population was detected. It was found that changes in the expression of epithelial (CKs and EpCAM) and mesenchymal markers were caused by changing the growth type of the tumor population. IFNα-2b inhibited migration of tumor cells to the suspension fraction and promoted an increase in expression of CK and EpCAM in 2D and 3D cell cultures, but only in the 3D culture was expression of vimentin increased. IFNα-2b caused an increase in CK and EpCAM expression by 50.5% and 47.8%, respectively, compared with the control in the 2D cell culture. In the 3D cell culture this increase was 33% and 34%, respectively, compared with the control. IFNα-2b stimulated the differentiation and inhibited the migrational ability of tumor cells in the early stages of breast cancer development.

Diosgenin induces genotoxic and mutagenic effects on HepG2 cells

Yam roots and other plants from Dioscorea genus have cultural, nutritional and economic importance to tropical and subtropical regions and have a great amount of diosgenin in its composition. In the present study the cytotoxic, genotoxic and mutagenic potential of diosgenin on HepG2 cells was investigated. Cytotoxicity was assessed using MTT and clonogenic assay. Genotoxic and mutagenic effects were performed using single cell gel electrophoresis and cytokinesis-block micronucleus assay, respectively. A reduction on cell viability was observed due to diosgenin treatment at concentrations higher than 30 μM. A genotoxic effect was shown by comet assay and CBMN. Besides, an increase in micronucleus frequency along with a significant cytostatic effect were observed. Diosgenin elicited DNA damage on HepG2 cells which could not be efficiently repaired contributing to the mutagenic effect observed. Those results suggest that diosgenin deleterious effect could take place through genetic instability, fact that affects the normal cell cycle, leading to cell's death.

Functioning of Fluorescent Proteins in Aggregates in Anthozoa Species and in Recombinant Artificial Models

Despite great advances in practical applications of fluorescent proteins (FPs), their natural function is poorly understood. FPs display complex spatio-temporal expression patterns in living Anthozoa coral polyps. Here we applied confocal microscopy, specifically, the fluorescence recovery after photobleaching (FRAP) technique to analyze intracellular localization and mobility of endogenous FPs in live tissues. We observed three distinct types of protein distributions in living tissues. One type of distribution, characteristic for Anemonia, Discosoma and Zoanthus, is free, highly mobile cytoplasmic localization. Another pattern is seen in FPs localized to numerous intracellular vesicles, observed in Clavularia. The third most intriguing type of intracellular localization is with respect to the spindle-shaped aggregates and lozenge crystals several micrometers in size observed in Zoanthus samples. No protein mobility within those structures was detected by FRAP. This finding encouraged us to develop artificial aggregating FPs. We constructed “trio-FPs” consisting of three tandem copies of tetrameric FPs and demonstrated that they form multiple bright foci upon expression in mammalian cells. High brightness of the aggregates is advantageous for early detection of weak promoter activities. Simultaneously, larger aggregates can induce significant cytostatic and cytotoxic effects and thus such tags are not suitable for long-term and high-level expression.

Synthesis of 2,6-Substituted 7-(Het)aryl-7-deazapurine Nucleobases (2,4-Disubstituted 5-(Het)aryl-pyrrolo[2,3-d]pyrimidines)

A series of 7-(het)aryl-7-deazapurine nucleobases (5-[(het)aryl]-2,4-disubstituted 7H-pyrrolo[2,3-d]pyrimidines) bearing NH2, OMe, SMe, or Me groups at position 6 and H, NH2, or Me at position 2 were prepared by the aqueous Suzuki–Miyaura cross-coupling reactions from SEM-protected 7-iodo-7-deazapurines with (het)arylboronic acids followed by deprotection. The 6-methoxy derivatives were further transformed into 7-deazahypoxanthines or 7-deazaguanines by O-demethylation reactions. Unlike their ribonucleoside counterparts, the 7-deazapurine nucleobases did not exert any significant cytostatic or antiviral effects.

Targeted TET oxidase activity through methyl-CpG-binding domain extensively suppresses cancer cell proliferation.

DNA methyltransferase (DNMT) inhibitors are epigenetic drugs used to treat myelodysplastic syndrome. They not only induce DNA demethylation but also have significant cytostatic and cytotoxic effects; however, the relationships between these characteristics have not been established yet due to the lack of a method to induce only DNA demethylation. Herein, we show that a fusion protein comprised of the methyl‐CpG‐binding domain (MBD) and the catalytic domain of Ten‐eleven translocation protein 1 (TET1‐CD) globally demethylates and upregulates a number of methylated genes. These upregulated genes frequently contained CpG islands (CGIs) within ± 1000 bp of the transcription start site (TSS). Interestingly, 65% of the genes upregulated fivefold or more by MBD‐TET1‐CDwt were also reactivated after treatment with a DNMT inhibitor, 5‐azacytidine (Aza‐CR), suggesting that gene reactivation by both methods primarily shares the same mechanism, DNA demethylation. In order to examine whether DNA demethylation affects the growth of cancer cells, we have established a tetracycline inducible system that can regulate the expression of MBD‐TET1‐CDwt in a prostate cancer cell line, LNCaP. The induction of MBD‐TET1‐CDwt demethylated and upregulated glutathione S‐transferase pi 1 (GSTP1), one of the hypermethylated genes in prostate cancer. In accordance with the reactivation of methylated genes, induction of MBD‐TET1‐CDwt extensively suppressed the growth of LNCaP cells through G1/S arrest. These results clearly indicate that TET oxidase activity recruited at methyl‐CpG sites through MBD induces reactivation of hypermethylated genes by DNA demethylation and allows us to analyze the effect of only global DNA demethylation in a wide variety of cancer cells.

Abstract 4432: Targeted TET oxidase activity through methyl-CpG binding domain extensively suppresses cancer cell proliferation

Abstract DNA demethylating agents are useful therapeutic drugs for human cancer; 5-aza-2’-deoxycytidine and 5-azacytidine for myelodysplastic syndrome are good examples. They not only induce DNA demethylation but also have significant cytostatic and cytotoxic effects. However, precise mechanisms for anticancer activity of these demethylating reagents have yet to be established, mainly due to the lack of method to induce only DNA demethylation. Here we show that a fusion protein comprising the methyl-CpG binding domain (MBD) and the catalytic domain of Ten-eleven translocation protein 1 (TET1-CD) globally demethylates and upregulates a number of methylated genes. Gene expression microarray analyses using human embryonic kidney cell line 293T indicate that cells expressing wild-type (wt) TET1 catalytic domain with MBD (MBD-TET1-CDwt) upregulates more genes than ones expressing TET1-CDwt without MBD (TET1-CDwt) or catalytically inactive TET1-CD mutant (mut) with MBD (MBD-TET1-CDmut) and their upregulated genes frequently contained CpG islands (CGIs) within ± 1,000-bp of the transcription start site (TSS). Interestingly, 65% of genes upregulated 5-fold or more by MBD-TET1-CDwt were also reactivated after treatment with 5-azacytidine, DNA demethylating agent. These results suggested a fact that gene reactivation by both methods was primarily based on DNA demethylation. In order to examine growth inhibitory effects of DNA demethylation to cancer cells, we utilized a tetracycline inducible system to regulate the expression of MBD-TET1-CDwt using a prostate cancer cell line. Induction of MBD-TET1-CDwt demethylated and upregulated the glutathione S-transferase pi 1 (GSTP1), one of the hypermethylated genes in prostate cancer. In accordance with reactivation of methylated genes, induction of MBD-TET1-CDwt extensively suppressed the growth of LNCaP cells. Flow cytometry analysis showed decreased cells in S phase and increased cells in G1 phase by MBD-TET1-CDwt expression. Our present results clearly indicate that TET oxidase activity recruited at methyl-CpG sites through MBD induces reactivation of hypermethylated genes by DNA demethylation, and that our methods allow us to analyze the effects of global DNA demethylation in a wide variety of cancer cells. Citation Format: Shinichi Fukushige, Yasuhiko Mizuguchi, Kanchan Chakma, Yuriko Saiki, Akira Horii. Targeted TET oxidase activity through methyl-CpG binding domain extensively suppresses cancer cell proliferation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4432.

Abstract 777: Effects of Src inhibitors and soy isoflavones on human prostate cancer cells

Abstract Background: Src family kinases are often over-expressed and highly active in solid tumors, including prostate cancer. This phenotype is associated with a poor prognosis partly because Src is a key factor in important signaling pathways involved in cell proliferation, angiogenesis, and initiation of metastasis. As a result, several promising small molecule targeting agents have been developed to prevent phosphorylation of key tyrosine residues that produce Src activation. However, these agents are not without side effects. The purpose of this study was to determine if adding a cytostatic soy isoflavone extract (ISF) to the treatment would produce optimal results at lower doses of the Src inhibitor. The ISF used in these studies has been shown to inhibit tumor growth in mice carrying human prostate cancer cell xenografts without causing toxicity to the host. Methods: Aggressive PC-3ML cells (a gift from A. Fatatis, Drexel University), and weakly tumorigenic LNCaP cells were treated for 24 hr with various concentrations of Src inhibitors (dasatinib, saracatinib), ISF (200 ug/ml, NovaSoy), or a combination. To evaluate effects on the metastatic potential of the cells, functional assays of cell growth and motility were performed, including those that assess clonogenic cell survival, cell cycle progression, and transwell migration and invasion activity. Results: Src inhibitors and ISF alone produced very little reduction in cell viability, but significant cytostatic effects, as determined by a reduction in clonogenicity, the number of cells able to form 50-cell colonies. This was likely due, in part, to changes in cell cycle progression. Both dasatinib and saracatinib caused an accumulation of cells in the G1 phase. As expected, ISF treatment resulted in higher numbers of cells in the G2/M phase. When the treatments were combined, cells exposed to dasatinib were observed to accumulate in both phases, with a significant decrease in S-phase cells. The results were not significant for saracatinib-treated cells. Exposure to either a Src inhibitor or ISF significantly reduced the migration of cells in a transwell chamber and their ability to invade through a Matrigel-coated 8 micron-pore membrane towards a chemoattractant (media containing 10% FBS). Dasatinib produced much greater effects, and at lower concentrations, than saracatinib. When combined with ISF, the effects were enhanced, particularly with dasatinib. Conclusions: In vitro studies suggested that combining a Src inhibitor and ISF resulted in greater inhibition of metastatic potential than either alone. This may indicate that including ISF in treatment regimens may allow a lower dose of the targeting agent to be used to achieve optimal response and also decrease toxicity. Citation Format: Lori P. Rice, Christine Pampo, Sharon Lepler, Dietmar W. Siemann. Effects of Src inhibitors and soy isoflavones on human prostate cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 777. doi:10.1158/1538-7445.AM2015-777

In vitro Assessments of Cytotoxic and Cytostatic Effects of Asparagus aphyllus, Crataegus aronia, and Ephedra alata in Monocultures and Co-Cultures of Hepg2 and THP-1-Derived Macrophages

Introduction: Herbal-based medicines are widely used for the prevention and treatments of diverse diseases especially in growing countries as well as many developed countries. Although some of herbal-based medicines have promising therapeutic properties, many of them remain untested and their safety and efficacy were not scientifically assessed. Based on knowledge from traditional Greco-Arab herbal medicine, this in vitro study aims to evaluate cytotoxic and cytostatic effects of three traditionally used anti-diabetic and anti-cancer medicinal plants. Out of the traditional medicinal plants, Asparagus aphyllus, Crataegus aronia, and Ephedra alata are widely used in many Mediterranean countries as natural remedies. Methods: Human THP-1- derived macrophages, HepG2 cells and their co-cultures were used in this in vitro study. Cells were treated for 24h (cytotoxic effects) and 72h (cytostatic effects) with increasing concentrations (0-1000 μg/ml) of water/ethanol extracts from Asparagus aphyllous (AA-extract), Crataegus aronia (CA-extract) and Ephedra alata (EA-extract). Cytotoxic and cytostatic effects were assessed using MTT assay and LDH assay. Results: No significant cytotoxic effects were seen with the three extracts up to concentration of 500 μg/ml. A slight cytotoxic effect was observed with CA-extractin HepG2 monocultures at concentrations higher than 500 μg/ml. Significant cytostatic effects were measured with CA-extract and EA-extract in monocultures and cocultures. The cytostatic activity of the extracts was more potent in co-cultures reaching IC50 of 178 μg/mL and 380 μg/mL for CA-extract and EA-extract, respectively. Conclusion: These results indicate that the traditionally known anti-cancer effects of CA-extract and EA-extract might be mediated in part through cytostatic effects.