Half-maximal Growth Inhibitory Concentrations Research Articles

Potential anti-proliferative activity of Salix mucronata and Triticum spelta plant extracts on liver and colorectal cancer cell lines

Cancer’s etiology is linked to oxidative stress. As a result, it's vital to find effective natural antioxidant remedies. Salix mucronata and Triticum spelta plant extracts were prepared using five different solvents and examined for their cytotoxicity against liver HepG2 cancer cell line. It was found that Salix mucronata ethanolic extract is high in antioxidant mediated anti-cancer activity. The functional constituents (phenolic and flavonoids) as well as preparation of different ethanolic concentrations used to study their properties that include DPPH, oxygen, hydroxyl, nitrogen radical scavenging activities, ferric reducing power and metal chelating activities. The MTT assay was used to determine antioxidant-mediated anti-cancer activity against human liver (HepG2) and colorectal (Caco-2) cancer cells to calculate the half-maximal growth inhibitory concentration (IC50). Moreover, flow cytometry analysis was used to quantify the apoptotic effect on the treated cancer cells. Additionally, qRTPCR of p53, BCL2, Cyclin D, MMP9 and VEGF were measured. Furthermore, HPLC was used to assess the most effective ingredients of the plant extract. Salix mucronata 50% ethanol extract had the highest polyphenolic content, anti-oxidant, and anti-proliferative activity. Salix mucronata increased the number of total apoptotic cells, and caused an upregulation of p53 gene expression by more than five folds and a downregulation of gene expression level of BCL2, Cyclin D, MMP9 and VEGF by more than five folds. Consequently, that could modulate oxidative stress and improve the effectiveness of cancer therapy. Results, also, showed that Triticum spelta ethanolic extract was less effective than Salix mucronata. Therefore, Salix mucronata ethanolic extract represents promising surrogate natural therapy for apoptosis-mediated cancer and recommended for further investigation using animal model.

MODL-05 Metronomic Intrathecal Delivery of CDK4/6 Inhibitors in Preclinical Models of Pediatric Brain Tumors

INTRODUCTION: CDK4/6 inhibitors have shown promise against central nervous system (CNS) tumors in vitro. This class of drugs relies on long-term exposure. Their use in early phase clinical studies in children with CNS tumors has defined dose limitations due to systemic toxicity. We have sought to circumvent these limitations in using CDK4/6 inhibitors for pediatric CNS tumors by first demonstrating enhanced efficiency with long-term administration and exploiting prolonged intrathecal delivery (IT). METHODS: Pediatric CNS tumor cell lines were used for cell viability assays: ATRT (BT-12, BT-16), CPC (CCHE-45), diffuse midline glioma (DIPG-XIII, HSJD-007), and medulloblastoma (DAOY); the assays were conducted at 24h, 72h, and 7d post-administration of CDK4/6 inhibitors (abemaciclib, palbociclib, ribociclib). Half maximal growth inhibitory concentrations (GI50) and areas under the curve (AUC) were compared for short-term (24h, 72h) and long-term (7d) dose-response curves. Toxicity with chronic IT administration was assessed using a neurobehavioral safety profile of 7-day continuous infusion of 2.5mM palbociclib (n = 5) into the mouse lateral ventricle compared with vehicle (n = 4). RESULTS: Our results demonstrate increased CDK4/6 inhibitor potency with longer administration. The greatest reductions in short-term to long-term GI50 were observed in ATRT, CPC, and DIPG across all inhibitors. The most pronounced time-dependent efficacy was observed with palbociclib for ATRT and abemaciclib for CPC and DIPG. AUCs significantly decreased (P < 0.05) with increasing drug exposure time across all inhibitors. 7-day intraventricular palbociclib infusion was equivalent in safety to PBS at doses ranging from 1,000 to 10,000-fold the in vitro GI50. CONCLUSIONS: The efficiency of CDK4/6 inhibitors in pediatric CNS tumors is enhanced with prolonged exposure. Long-term IT administration can achieve high CNS doses without associated systemic toxicities. Translational efforts using a metronomic IT strategy are logical to explore for pediatric CNS tumors which have potential for a leptomeningeal disease pattern.

Preparation, Characterization and Anticancer Activity of Chitosan Prepared from the American Cockroach, Periplaneta americana

The exoskeleton of a cockroach plays a major role in protecting it from pathogenic microbes. It is well known that the main component of the exoskeleton is the chitin which is considered the source of chitosan. This study aimed at investigating the anticancer activity of chitosan prepared from the American cockroach. Chitosan was prepared and characterized with solubility in 1% acetic acid and Fourier transform infrared spectroscopy (FT-IR) analysis. The cytotoxicity of chitosan was investigated against Hepatoblastoma (HepG2) and Breast cancer (MCF7) cell lines by the MTT assay. The cytotoxicity has a positive relationship with the chitosan concentration. The half-maximal growth inhibitory concentrations (IC50) were 329 and 195 µg/ml with HepG2 and MCF7, respectively. Therefore, Chitosan may be a potential natural compound for the treatment of human hepatoblastoma (HepG2) and breast cancer (MCF7).

Comparative in vitro and in silico characterization of anticancer compounds piceatannol, biochanin-A and resveratrol on breast cancer cells

Background: Biochanin-A and Piceatannol are phytochemical constituents extracted from Sophora interrupta. Although both the compounds were isolated from a single plant, these compounds were not compared against anticancer activity. Objective: A systematic comparative analysis of biochanin-A, piceatannol, and resveratrol was performed to investigate cancer cell viability, motility, metabolic changes in Michigan Cancer Foundation-7 breast cancer cells, and structure compound interaction with the vascular endothelial growth factor (VEGF) receptors were studied. Materials and Methods: Cancer cell viability was studied using 3 (4, 5 dimethyl thiazol 2yl) 2, 5 diphenyltetrazo- lium bromide and acridine orange (AO)/ethidium bromide (EtBr) assay. The wound-healing assay was performed by measuring cell migration from the scratch area. Metabolic changes of the compounds in culture conditions were recorded using Fourier-transform infrared (FT-IR) spectroscopy. Molecular docking and dynamic simulations were performed using Schrodinger software. Results: Our results showed that the half-maximal growth inhibitory concentration for biochanin-A is 150 μM/ml and piceatannol and resveratrol showed 150 μM/ml, which is evident from the uptake of AO and EtBr dyes by live/dead cells. Moreover, drug-treated cells were unable to fill the cleared area from the scratch area, which suggests that all compounds effectively inhibit cell motility. FT-IR fingerprint showed a marked difference in the percentage of transition and dynamic structural changes between untreated and treated samples. Strong hydrogen-bond interaction with VEGF receptor-1 (VEGFR1) and VEGFR2 proteins and their interactions were stable throughout the simulation period. Moreover, these compounds inhibited sprouting of a new blood vessel from the chicken aorta and microvessels formation in the in ovo chorioallantoic membrane assay. Conclusion: Taken together, we conclude that anticancer and anti-angiogenic activity, structure-function relationship of biochanin-A is like well-known anticancer compound resveratrol and its metabolic product piceatannol in breast cancer cells.

Modes of cell death induced by tetrahydroisoquinoline-based analogs in MDA-MB-231 breast and A549 lung cancer cell lines.

BackgroundA and B rings of the steroidal microtubule disruptor, 2-methoxyestradiol, and its analogs can be mimicked with a tetrahydroisoquinoline (THIQ) core. THIQs are cytotoxic agents with potential anticancer activities. The aim of this in vitro study was to investigate the modes of cell death induced by four nonsteroidal THIQ-based analogs, such as STX 2895, STX 3329, STX 3451 and STX 3450, on MDA-MB-231 metastatic breast and A549 epithelial lung carcinoma cells.Materials and methodsCytotoxicity studies determined the half-maximal growth inhibitory concentration of the analogs to be at nanomolar concentrations without the induction of necrosis. Light and fluorescent microscopy determined that compounds caused microtubule depolymerization and displayed morphological hallmarks of apoptosis.ResultsFlow cytometric analyses confirmed apoptosis induction as well as an increased G2/M phase on cell cycle analysis. Furthermore, intrinsic pathway signaling was implicated due to increased cytochrome c release and a decrease in mitochondrial transmembrane potential. Potential involvement of autophagy was observed due to increased acidic vacuole formation and increased aggresome activation factor.ConclusionThus, it can be concluded that these four THIQ-based analogs exert anti-proliferative and antimitotic effects, induce apoptosis and involve autophagic processes. Further investigation into the efficacy of these potential anticancer drugs will be conducted in vitro and in vivo.

Effects of natural phenolic compound carvacrol on the human gastric adenocarcinoma (AGS) cells in vitro.

Gastric cancer (GC) is the most common cause of morbidity and mortality because of cancer. Medicinal plants containing polyphenolic compounds have gained importance in anticancer treatment. In this context, carvacrol is a main component of many plants in the family Lamiaceae that are frequently used in folk medicine and a good candidate to investigate for GC treatment. The present study aimed to explore the cytotoxic, genotoxic, apoptotic, and reactive oxygen species (ROS)-generating effects of carvacrol on gastric adenocarcinoma in vitro. For these purposes, human gastric adenocarcinoma (AGS) cells were used and analyzed after 24 h of exposure to carvacrol with different concentrations. The cytotoxicity, ROS generation, glutathione (GSH) level, and genotoxicity were investigated by the ATP cell viability assay, 2',7'-dichlorodihydrofluorescein-diacetate assay, GSH/GSSG-Glo assay, and comet assay, respectively. Apoptosis induction was detected by acridine orange/ethidium bromide staining and western blotting at below the half-maximal growth inhibitory concentration value. Carvacrol showed cytotoxic, genotoxic, apoptotic, ROS generating, and GSH-reducing effects on AGS cells in a dose-dependent manner. There was a close negative relationship between cell viability and ROS level. Carvacrol inhibited the proliferation of AGS cells, suggesting that it could be a novel and strong anticancer agent against the human gastric adenocarcinoma. These results support the interest of natural diet components in the development of therapeutic products for diseases.

P10. Radiosensitization of breast adenocarcinoma cells by a novel estrone analogue is dependent on reactive oxygen species signalling

Introduction and aim The novel antimitotic drug 2-ethyl-3-O-sulpamoyl-estra-1,3,5(10),15-tetraen-17-ol (ESE-15-ol) is an in silico-designed 17-β-estradiol analogue that induces apoptosis and disregulates autophagy in various neoplastic cell lines. The compound alters microtubule dynamics inducing both intrinsic and extrinsic death pathways. The hypothesis was formed that pre-exposure to the compound would sensitize breast adenocarcinoma (MDA-MB-231) cells to gamma radiation, in pathways dependent on reactive oxygen species (ROS) generation. Methods and materials The hypothesis was tested by inhibiting ROS formation with N-acetyl-cysteine (NAC). The half-maximal growth-inhibitory concentration ( IG 50 ) of ESE-15-ol after 24 h was determined with and without NAC via spectrophotometric quantification of crystal violet. MDA-MB-231 cells were presensitized with half the IG 50 concentrations prior to 6 Gy radiation. Morphological studies were done using polarized-optical transmitted light differential interference light microscopy and confocal microscopy using anti-α tubulin antibodies and Mitotracker® mitochondrial staining. Quantitative studies were completed using flow cytometry to detect apoptosis induction, loss of mitochondrial membrane potential and reactive oxygen species production, as well as to analyze cell cycle progression and mitochondrial signal transduction pathways. Results The IG 50 of ESE-15-ol was determined at a concentration of 0.18 μM after a 24-h exposure. Results obtained from the morphological studies indicated that incubation with NAC reduced apoptotic cell death despite microtubule depolarization after ESE-15-ol presensitization prior to gamma radiation. Flow cytometric quantification indicated a reduction in apoptosis, mitochondrial membrane depolarization, as well as in reactive oxygen species when samples were concurrently undergoing antioxidant treatment. Conclusion This in vitro study revealed that inhibition of reactive oxygen species signalling resulted in a decreased effectiveness of ESE-15-ol to presensitize MDA-MB-231 cells to gamma radiation. Thus the hypothesis was accepted. Further quantification of ROS on autophagy induction, as well as its role in the radiosensitization mechanisms, will be carried out in vitro and in vivo.

Ketorolac salt is a newly discovered DDX3 inhibitor to treat oral cancer

DDX3 belongs to DEAD box RNA helicase family and is involved in the progression of several types of cancer. In this work, we employed a High Throughput Virtual screening approach to identify bioactive compounds against DDX3 from ZINC natural database. Ketorolac salt was selected based on its binding free energy less than or equals to −5 Kcal/mol with reference to existing synthetic DDX3 inhibitors and strong hydrogen bond interactions as similar to crystallized DDX3 protein (2I4I). The anti-cancer activity of Ketorolac salt against DDX3 was tested using oral squamous cell carcinoma (OSCC) cell lines. This compound significantly down regulated the expression of DDX3 in human OSCC line (H357) and the half maximal growth inhibitory concentration (IC50) of Ketorolac salt in H357 cell line is 2.6 µM. Ketorolac salt also inhibited the ATP hydrolysis by directly interacting with DDX3. More importantly, we observed decreased number of neoplastic tongue lesions and reduced lesion severity in Ketorolac salt treated groups in a carcinogen induced tongue tumor mouse model. Taken together, our result demonstrates that Ketorolac salt is a newly discovered bioactive compound against DDX3 and this compound can be used as an ideal drug candidate to treat DDX3 associated oral cancer.

Karwinaphthopyranones from the fruits of Karwinskia parvifolia and their cytotoxic activities.

The new 2-acetyl-8-methoxynaphthol (1) and five new "dimeric" napthopyranones, karwinaphthopyranones A1 and A2 (2 and 3) and karwinaphthopyranones B1-B3 (4-6), possessing a methoxy group at C-5', were isolated together with four other known compounds from the dried fruits of Karwinskia parvifolia. The structures of compounds 2-6 were determined by spectroscopic data interpretation. Cell culture assays showed that some of these compounds possess antiproliferative activities in representative human cancer cell lines, with half-maximal growth inhibitory concentrations in the micromolar range.

Anti-tumor activities of Au(I) complexed with bisphosphines in HL-60 cells

We found that Au(I) complexed with 2,3-bis(tert-butyl(methyl)phosphino) quinoxaline (10) was a potent anti-tumor agent (half-maximal growth inhibitory concentration, GI50=0.87μM) with broad anti-tumor activity. In particular, the activity of complex 10 was high in tumor cell lines derived from the colon and ovary. Treatment with complex 10 resulted in the apoptosis of HL-60 cells. The ligand for the preparation of complex 10 is commercially available implying that complex 10 might be a good drug candidate for cancer therapy.

Abstract 2257: Thailanstatins: New pre-mRNA splicing inhibitors and potent antiproliferative agents discovered from Burkholderia Thailandensis MSMB43.

Abstract Natural products have been traditionally sought from actinomycetes, filamentous fungi and medicinal plants. Gram-negative bacterial species, such as Burkholderia, Chromobacterium, Lysobacter, Pseudomonas and Xenorhabdus, however, have recently captured attention as new sources of diverse bioactive natural products. Mining the genome sequence of B. thailandensis MSMB43 revealed a cryptic biosynthetic gene cluster highly resembling that of FR901,464, a prototype pre-mRNA splicing inhibitor produced by Pseudomonas sp. No. 2663. Transcriptioal analysis identified a cultivation condition in which a representive gene of the cryptic gene cluster is adequately expressed. Consequently, three new compounds, thailanstatins A, B and C, were isolated from the fermentation broth of B. thailandensis MSMB43 through natural product chemistry. Thailanstatins belong to the FR901,464-family of microbial products biosynthesized by a hybrid polyketide synthease-nonribosomal peptide synthetase pathway. They have an oveall structural similarity with FR901,464, but feature an extra acetic acid unit which endows the compouds with a significantly higher stability than FR901,464 under physiologically relevant conditions. In vitro assays showed that thailanstatins inhibit pre-mRNA splicing as potently as FR901,464, with half-maximal inhibitory concentrations in the single to sub μM range, causing pre-mRNA to accumulate and preventing the production of mRNA and splicing intermediates. In vitro cell culture assays indicated that thailanstatins also possess potent antiproliferative activities in representative human cancer cell lines, with half-maximal growth inhibitory concentrations in the single nM range (Table 1). This work provides new chemical entities for research and development, and validates the Burkholderia species as an exciting new source of bioactive natural products. Table 1.Antiproliferative activities of compounds in human cancer cell lines (GI50 in nM).CompoundDU-145 (prostate cancer)H232A (non-small cell lung cancer)MDA-MB235 (triple-negative breast cancer)SKOV-3 (ovarian cancer)Thailanstatin A1.11±0.022.26±0.172.58±0.112.69±0.37Thailanstatin B3.00±0.922.50±0.066.22±1.674.94±1.76Thailanstatin C2.98±0.903.67±0.538.82±2.205.57±2.01FR901,4641.05±0.021.94±0.242.10±0.191.06±0.01 Citation Format: Xiangyang Liu, Sreya Biswas, Michael G. Berg, Christopher M. Antapli, Feng Xie, Qi Wang, Man-Cheng Tang, Gong-Li Tang, Lixin Zhang, Gideon Dreyfuss, Yi-Qiang Cheng. Thailanstatins: New pre-mRNA splicing inhibitors and potent antiproliferative agents discovered from Burkholderia Thailandensis MSMB43. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2257. doi:10.1158/1538-7445.AM2013-2257

Genomics-Guided Discovery of Thailanstatins A, B, and C As Pre-mRNA Splicing Inhibitors and Antiproliferative Agents from Burkholderia thailandensis MSMB43

Mining the genome sequence of Burkholderia thailandensis MSMB43 revealed a cryptic biosynthetic gene cluster resembling that of FR901464 (4), a prototype spliceosome inhibitor produced by Pseudomonas sp. No. 2663. Transcriptional analysis revealed a cultivation condition in which a regulatory gene of the cryptic gene cluster is adequately expressed. Consequently, three new compounds, named thailanstatins A (1), B (2), and C (3), were isolated from the fermentation broth of B. thailandensis MSMB43. Thailanstatins are proposed to be biosynthesized by a hybrid polyketide synthase-nonribosomal peptide synthetase pathway. They differ from 4 by lacking an unstable hydroxyl group and by having an extra carboxyl moiety; those differences endow thailanstatins with a significantly greater stability than 4 as tested in phosphate buffer at pH 7.4. In vitro assays showed that thailanstatins inhibit pre-mRNA splicing as potently as 4, with half-maximal inhibitory concentrations in the single to sub-μM range. Cell culture assays indicated that thailanstatins also possess potent antiproliferative activities in representative human cancer cell lines, with half-maximal growth inhibitory concentrations in the single nM range. This work provides new chemical entities for research and development and new structure-activity information for chemical optimization of related spliceosome inhibitors.

Metaphase arrest and delay in cell cycle kinetics of root apical meristems and mouse bone marrow cells treated with leaf aqueous extract of Clerodendrum viscosum Vent

To study cell cycle delay and metaphase arresting activity of leaf aqueous extract of Clerodendrum viscosum Vent. (LAECV) in root apical meristems and mouse bone marrow cells. Cell cycle delay and metaphase arresting activities of LAECV were analysed, in root apical meristems of onion and wheat, and in mouse bone marrow cells, by scoring mitotic index, metaphase frequency and transition of cells from metaphase to anaphase. Colchicine was used as the standard metaphase arresting drug. Phytochemicals present in LAECV were detected and their phytotoxic activity was evaluated by analysing green-gram (Vigna radiata) seedling's root growth retardation and branch root swelling phenomenon. LAECV treatment resulted in dose-dependent root growth retardation of green-gram seedling root length (P < 0.01) and half maximal growth inhibitory concentration (IC(50) ) of LAECV was 0.87 mg/ml at 144 h. In onion and wheat root meristem cells the mitotic index decreased, metaphase frequency increased and transition from metaphase to anaphase reduced. Experimentation with mouse bone marrow cells indicated that LAECV induced metaphase arrest (164.3% increase in arrested metaphases per 300 mg/kg body weight, over 2.5 h). Phytochemicals like carbohydrates, glycosides, saponins, terpenoids, triterpenoids, tannins and trace amounts of alkaloids were detected in LAECV. It may be said that LAECV contains mitostatic and metaphase arresting components that are able to induce significant metaphase arrest in root apical meristems and also in mouse bone marrow cells.

Tolfenamic acid inhibits esophageal cancer through repression of specificity proteins and c-Met

The non-steroidal anti-inflammatory drug tolfenamic acid (TA) inhibits proliferation of SEG-1 and BIC-1 esophageal cancer cells with half-maximal growth inhibitory concentration values of 36 and 48 muM, respectively. TA also increased Annexin V staining in both cell lines, indicative of proapoptotic activity. Treatment of SEG-1 and BIC-1 cells with TA for up to 72 h decreased expression of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 and this was accompanied by decreased expression of the well-characterized Sp-regulated genes cyclin D1, vascular endothelial growth factor and survivin. TA also decreased hepatocyte growth factor receptor, (c-Met), a receptor tyrosine kinase that is overexpressed in esophageal cancer cells and tumors and is an important drug target. Knockdown of Sp1, Sp3 and Sp4 by RNA interference in SEG-1 and BIC-1 cells also decreased c-Met expression, demonstrating that c-Met is an Sp-regulated gene in esophageal cancer cells. Sp1 was overexpressed in esophageal cancer cells and tumors and increased Sp1 staining was observed in esophageal tumors from patients. TA (20 mg/kg/day) also decreased tumor growth and weight in athymic nude mice bearing SEG-1 cells as xenografts and this was accompanied by increased apoptosis and decreased Sp1 and c-Met staining in tumors from treated mice. Thus, TA-dependent downregulation of Sp transcription factors and c-Met defines a novel chemotherapeutic approach for treatment of esophageal cancer.

Cytotoxic effect of the cyclosporin PSC 833 in multidrug-resistant leukaemia cells with increased expression of P-glycoprotein

Multidrug resistance (MDR) to anti-cancer agents is frequently associated with overexpression of the drug efflux transporter P-glycoprotein (Pgp) in cancer cells, ensuing drug expulsion and maintenance of tolerable intracellular levels of certain cytotoxic drugs. Pgp may also be present in normal tissue, providing protection against toxic substances, but the physiological role of Pgp is not fully understood. Recently, it was shown that Pgp also takes part in the transport of certain growth-regulating cytokines (Drach et al, 1996; Raghu et al, 1996). Therefore, we studied the effect of the highly potent Pgp inhibitor PSC 833 on proliferation of three pairs of MDR and parental human cell lines (HB8065 hepatoma cells, KG1a and K562 leukaemia cells). The MDR phenotypes were characterized by Pgp overexpression, which was demonstrated by flow cytometry using the anti-Pgp antibody MRK16. Electronic cell counting of 72-96 h cultures revealed a dose-dependent antiproliferative effect of PSC 833 in the resistant KG1a/200 and K562/150 cells. The half-maximal growth inhibitory concentrations (GI50) were 0.2 microM and 0.7 microM respectively. Exposure to PSC 833 induced cell death by apoptosis in both cell types, as revealed by flow cytometry and detection of 3'-hydroxy ends of DNA (the result of DNA fragmentation associated with apoptosis), by terminal transferase-mediated dUTP-biotin nick end-labelling (TUNEL). Similar effects were not found in the hepatoma cell lines or the parental leukaemia lines. These results demonstrated a discriminating cytotoxicity of PSC 833 in two human leukaemia MDR variants, representing a possible therapeutic indication which warrants consideration during the ongoing clinical evaluation of this drug.