Jelena Dinić

Diarylheptanoids from the bark of black alder inhibit the growth of sensitive and multi-drug resistant non-small cell lung carcinoma cells.

An extended study of minor diarylheptanoids from the bark of black alder has resulted in the isolation of twenty diarylheptanoids, ten of which have not previously been reported (14-18, 20-24). The structures and configurations of all compounds were elucidated by NMR, HRESIMS, UV, IR, and CD. The anti-cancer potency of twenty diarylheptanoids and four previously isolated compounds (7, 10, 12, 13) was investigated in human non-small cell lung carcinoma cell lines (sensitive and multi-drug resistant variants) as well as in normal human keratinocytes. Diarylheptanoids with a p-coumaroyl group, 14 and 18, platyphylloside (1), platyphyllonol-5-O-β-D-xylopyranoside (2), alnuside B (4) and hirsutenone (9) exhibited strong anti-cancer activity, considerably higher than diarylheptanoid curcumin, which served as a positive control. Compounds 4, 9, 14, and 18 displayed significant selectivity towards the cancer cells. Structure/activity analysis of twenty-four closely related diarylheptanoids revealed a high dependence of cytotoxic action on the presence of a carbonyl group at C-3. Substitution of a heptane chain on C-5 and a number of hydroxyl groups in the aromatic rings also emerged as a significant structural feature that influenced their cytotoxic potential.

Discovery of 14-3-3 protein-protein interaction inhibitors that sensitize multidrug-resistant cancer cells to doxorubicin and the Akt inhibitor GSK690693.

14‐3‐3 is a family of highly conserved adapter proteins that is attracting much interest among medicinal chemists. Small‐molecule inhibitors of 14‐3‐3 protein–protein interactions (PPIs) are in high demand, both as tools to increase our understanding of 14‐3‐3 actions in human diseases and as leads to develop innovative therapeutic agents. Herein we present the discovery of novel 14‐3‐3 PPI inhibitors through a multidisciplinary strategy combining molecular modeling, organic synthesis, image‐based high‐content analysis of reporter cells, and in vitro assays using cancer cells. Notably, the two most active compounds promoted the translocation of c‐Abl and FOXO pro‐apoptotic factors into the nucleus and sensitized multidrug‐resistant cancer cells to apoptotic inducers such as doxorubicin and the pan‐Akt inhibitor GSK690693, thus becoming valuable lead candidates for further optimization. Our results emphasize the possible role of 14‐3‐3 PPI inhibitors in anticancer combination therapies.

New Approaches With Natural Product Drugs for Overcoming Multidrug Resistance in Cancer.

Resistance to chemotherapeutic drugs is one of the main obstacles to effective cancer treatment. Multidrug resistance (MDR) is defined as resistance to structurally and/or functionally unrelated drugs, and has been extensively investigated for the last three decades. There are two types of MDR: intrinsic and acquired. Tumor microenvironment selection pressure leads to the development of intrinsic MDR, while acquired resistance is a consequence of the administered chemotherapy. A central issue in chemotherapy failure is the existence of heterogeneous populations of cancer cells within one patient and patient-to-patient variability within each type of cancer. Numerous genes and pathways contribute to the development of MDR in cancer. Point mutations, gene amplification or other genetic or epigenetic changes all affect biological functions and may lead to the occurrence of MDR phenotype. Similar to the characteristics of cancerogenesis, the main features of MDR include abnormal tumor vasculature, regions of hypoxia, aerobic glycolysis, and a lower susceptibility to apoptosis. In order to achieve a lethal effect on cancer cells, drugs need to reach their intracellular target molecules. The overexpression of the efflux transporter P-glycoprotein (P-gp) in MDR cancer cells leads to decreased uptake of the drug and intracellular drug accumulation, minimising drug-target interactions. New agents being or inspired by natural products that successfully target these mechanisms are the main subject of this review. Two key approaches in combating MDR in cancer are discussed (i) finding agents that preserve cytotoxicity toward MDR cancer cells; (ii) developing compounds that restore the cytotoxic activity of classic anticancer drugs.

MTHFR C677T polymorphism in chronic pancreatitis and pancreatic adenocarcinoma

Chronic pancreatitis and pancreatic adenocarcinoma are extensively studied as common and potentially lethal disorders. However, their causes and genetic background in most cases remain unclear. The C677T polymorphism in 5′,10′‐methylenetetrahydrofolate reductase (MTHFR) gene may modulate the risk of pancreatic disorders. In this study, we tested whether MTHFR C677T polymorphism is associated with chronic pancreatitis and pancreatic adenocarcinoma in the Serbian population. DNA was extracted from blood samples of 51 chronic pancreatitis patients, 21 pancreatic adenocarcinoma patients, and a control group consisting of 50 healthy smokers. The MTHFR C677T polymorphism was analyzed by polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) technique. Although, no statistically significant differences were observed in the distribution of MTHFR genotype or allele frequencies between patients and control groups, the results showed an increased frequency of homozygotes for MTHFR C677T polymorphism in chronic pancreatitis patients (14%) and a decreased frequency in pancreatic adenocarcinoma patients (5%) in comparison to the control group (8%). We speculate that the MTHFR C677T polymorphism could act as a possible risk factor for chronic pancreatitis and a possible protective factor in pancreatic adenocarcinoma. This observation needs further investigation in prospective studies on a larger number of patients, in which the effect of other genetic and environmental factors should also be taken into consideration. Copyright

Chemo-protective and regenerative effects of diarylheptanoids from the bark of black alder (Alnus glutinosa) in human normal keratinocytes.

Medicinal plants are recognized from ancient times as a source of diverse therapeutic agents and many of them are used as dietary supplements. Comprehensive approaches are needed that would identify bioactive components with evident activity against specific indications and provide a better link between science (ethno-botany, chemistry, biology and pharmacology) and market. Recently, the bark of black alder (Alnus glutinosa) appeared at market in the form of food supplement for treatment of different skin conditions. This study aimed to evaluate protective effects of two diarylheptanoids isolated from the bark of black alder: platyphylloside, 5(S)-1,7-di(4-hydroxyphenyl)-3-heptanone-5-O-β-D-glucopyranoside (1) and its newly discovered analog 5(S)-1,7-di(4-hydroxyphenyl)-5-O-β-D-[6-(E-p-coumaroylglucopyranosyl)]heptane-3-one (2) towards doxorubicin damaging activity. To that end, we employed HaCaT cells, non-cancerous human keratinocytes commonly used for skin regenerative studies. Diarylheptanoids significantly antagonized the effects of doxorubicin by lowering the sensitivity of HaCaT cells to this drug. Compound 2 prevented doxorubicin-induced cell death by activating autophagy. Both 1 and 2 protected HaCaT cells against doxorubicin-induced DNA damage. They significantly promoted migration and affected F-actin distribution. These results indicate that chemo-protective effects of diarylheptanoids may occur at multiple subcellular levels. Therefore, diarylheptanoids 1 and 2 could be considered as protective agents for non-cancerous dividing cells during chemotherapy.

Antioxidative Activity of Diarylheptanoids from the Bark of Black Alder (Alnus glutinosa) and Their Interaction with Anticancer Drugs

Diarylheptanoids belong to polyphenols, a group of plant secondary metabolites with multiple biological properties. Many of them display antioxidative, cytotoxic, or anticancer actions and are increasingly recognized as potential therapeutic agents. The aim of this study was to evaluate antioxidant and cytoprotective activity of two diarylheptanoids: platyphylloside 5(S)-1,7-di(4-hydroxyphenyl)-3-heptanone-5-O-β-D-glucopyranoside (1) and its newly discovered analog 5(S)-1,7-di(4-hydroxyphenyl)-5-O-β-D-[6-(E-p-coumaroylglucopyranosyl)]heptane-3-one (2), both isolated from the bark of black alder (Alnus glutinosa). To that end, we have employed a cancer cell line (NCI-H460), normal human keratinocytes (HaCaT), and peripheral blood mononuclear cells. The effects on cell growth were assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay. Cell death was examined by annexin V/propidium iodide staining on a flow cytometer. Reactive oxygen species production was examined by dihydroethidium staining. Mitochondrial structure and doxorubicin localization were visualized by fluorescent microscopy. Gene expression of manganese superoxide dismutase and hypoxia-inducible factor-1α was determined by reverse transcription polymerase chain reaction. Diarylheptanoids antagonized the effects of either doxorubicin or cisplatin, significantly increasing their IC50 values in normal cells. Diarylheptanoid 1 induced the retention of doxorubicin in cytoplasm and reduced mitochondrial fragmentation associated with doxorubicin application. Diarylheptanoid 2 reduced the reactive oxygen species production induced by cisplatin. Both compounds increased the messenger ribonucleic acid expression of enzymes involved in reactive oxygen species elimination (manganese superoxide dismutase and hypoxia-inducible factor-1α). These results indicate that neutralization of reactive oxygen species is an important mechanism of diarylheptanoid action, although these compounds exert a considerable anticancer effect. Therefore, these compounds may serve as protectors of normal cells during chemotherapy without significantly diminishing the effect of the applied chemotherapeutic.

Development of resistance to antiglioma agents in rat C6 cells caused collateral sensitivity to doxorubicin

Chemoresistance is a severe limitation to glioblastoma (GBM) therapy and there is a strong need to understand the underlying mechanisms that determine its response to different chemotherapeutics. Therefore, we induced resistance in C6 rat glioma cell line, which considerably resembles the characteristics of human GBM. The resistant phenotype was developed by 3-bis (2-chloroethyl)-1-nitrosourea (BCNU), one of the most commonly used therapeutic drug in the course of GBM treatment. After confirmation of the cross-resistance to cisplatin (CPt) and temozolomide (TMZ) in newly established RC6 cell line, we examined cell death induction and DNA damage by these drugs. Resistance to apoptosis and deficiency in forming DNA double-strand breaks was followed by significant decrease in the mRNA expression of pro-apoptotic and anti-apoptotic genes. The development of drug resistance was associated with significant increase in reactive oxygen species (ROS) and decrease in oxidized to reduced gluthatione ratio in RC6 cell line indicating a reduced level of oxidative stress. The mRNA expression levels of manganese superoxid dismutase (MnSOD), inducible nitric oxide synthase (iNOS) and gluthatione peroxidase (GPx) were increased while hypoxia-inducible factor 1-α (HIF-1α) was decreased in RC6 compared to C6 cells. This was in line with obtained changes in ROS content and increased antioxidative capacity of RC6 cells. Importantly, RC6 cells demonstrated collateral sensitivity to doxorubicin (DOX). The analysis of this phenomenon revealed increased accumulation of DOX in RC6 cells due to their adaptation to high ROS content and acidification of cytoplasm. In conclusion, newly established RC6 rat glioma cell line could be used as a starting material for the development of allogenic animal model and preclinical evaluation of new antiglioma agents. Collateral sensitivity to DOX obtained after BCNU treatment may prompt new studies aimed to find efficient delivery of DOX to the glioma site in brain.

Resistance to DNA Damaging agents produced invasive phenotype of rat glioma cells-characterization of a new in vivo model

Chemoresistance and invasion properties are severe limitations to efficient glioma therapy. Therefore, development of glioma in vivo models that more accurately resemble the situation observed in patients emerges. Previously, we established RC6 rat glioma cell line resistant to DNA damaging agents including antiglioma approved therapies such as 3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and temozolomide (TMZ). Herein, we evaluated the invasiveness of RC6 cells in vitro and in a new orthotopic animal model. For comparison, we used C6 cells from which RC6 cells originated. Differences in cell growth properties were assessed by real-time cell analyzer. Cells’ invasive potential in vitro was studied in fluorescently labeled gelatin and by formation of multicellular spheroids in hydrogel. For animal studies, fluorescently labeled cells were inoculated into adult male Wistar rat brains. Consecutive coronal and sagittal brain sections were analyzed 10 and 25 days post-inoculation, while rats’ behavior was recorded during three days in the open field test starting from 25th day post-inoculation. We demonstrated that development of chemoresistance induced invasive phenotype of RC6 cells with significant behavioral impediments implying usefulness of orthotopic RC6 glioma allograft in preclinical studies for the examination of new approaches to counteract both chemoresistance and invasion of glioma cells.

Transfer of Drug Resistance Characteristics Between Cancer Cell Subpopulations: A Study Using Simple Mathematical Models

Resistance to chemotherapy is a major cause of cancer treatment failure. The processes of resistance induction and selection of resistant cells (due to the over-expression of the membrane transporter P-glycoprotein, P-gp) are well documented in the literature, and a number of mathematical models have been developed. However, another process of transfer of resistant characteristics is less well known and has received little attention in the mathematical literature. In this paper, we discuss the potential of simple mathematical models to describe the process of resistance transfer, specifically P-gp transfer, in mixtures of resistant and sensitive tumor cell populations. Two different biological hypotheses for P-gp transfer are explored: (1) exchange through physical cell–cell connections and (2) through microvessicles released to the culture medium. Two models are developed which fit very well the observed population growth dynamics. The potential and limitations of these simple “global” models to describe the aforementioned biological processes involved are discussed on the basis of the results obtained.

Targeting autophagy to modulate cell survival: a comparative analysis in cancer, normal and embryonic cells

AbstractAutophagy is linked to multiple cancer-related signaling pathways, and represents a defense mechanism for cancer cells under therapeutic stress. The crosstalk between apoptosis and autophagy is essential for both tumorigenesis and embryonic development. We studied the influence of autophagy on cell survival in pro-apoptotic conditions induced by anticancer drugs in three model systems: human cancer cells (NCI-H460, COR-L23 and U87), human normal cells (HaCaT and MRC-5) and zebrafish embryos (Danio rerio). Autophagy induction with AZD2014 and tamoxifen antagonized the pro-apoptotic effect of chemotherapeutics doxorubicin and cisplatin in cell lines, while autophagy inhibition by wortmannin and chloroquine synergized the action of both anticancer agents. This effect was further verified by assessing cleaved caspase-3 and PARP-1 levels. Autophagy inhibitors significantly increased both apoptotic markers when applied in combination with doxorubicin while autophagy inducers had the opposite effect. In a similar manner, autophagy induction in zebrafish embryos prevented cisplatin-induced apoptosis in the tail region while autophagy inhibition increased cell death in the tail and retina of cisplatin-treated animals. Autophagy modulation with direct inhibitors of the PI3kinase/Akt/mTOR pathway (AZD2014 and wortmannin) triggered the cellular response to anticancer drugs more effectively in NCI-H460 and zebrafish embryonic models compared to HaCaT suggesting that these modulators are selective towards rapidly proliferating cells. Therefore, evaluating the autophagic properties of chemotherapeutics could help determine more accurately the fate of different cell types under treatment. Our study underlines the importance of testing autophagic activity of potential anticancer agents in a comparative approach to develop more rational anticancer therapeutic strategies.