Plants from Northwestern Anatolia Display Selective Cytotoxicity and Induce Mitotic Catastrophe: A Study on Anticancer and Genotoxic Activities.

Anatolia is rich in floristic diversity with a high rate of endemism. Eight plant species from northwestern Anatolia were evaluated for their anti-growth properties in two malignant (MCF-7 and MDA-MB-231) and a non-malignant (MCF-10A) breast cell lines. The two most active extracts, Achillea multifida (AME) and Astragalus sibthorpianus (ASE), induced apoptotic cell death in all cell lines. The major phenolic compounds in AME were identified as chlorogenic acid, and catechins in ASE. ASE displayed selective cytotoxicity against breast cancer cells, with DNA damage repair in non-malignant cells contributing to its selectivity. Conversely, AME induced DNA damage in a time-dependent manner and displayed a dual dose-dependent biological activity, resulting in mitotic catastrophe and apoptosis at different doses. Most plant species exhibited moderate to strong cytotoxicity, highlighting their medicinal and economic potential and the need for their protection.

Toxicity assessment of Hypericum olympicum subsp. olympicum L. on human lymphocytes and breast cancer cell lines.

There is a limited number of studies about the constituents of Hypericum olympicum subsp. olympicum and its genotoxic and cytotoxic potency. We examined the possible antigenotoxic/genotoxic properties of methanolic extract of H. olympicum subsp. olympicum (HOE) on human lymphocytes by employing sister chromatid exchange, micronucleus and comet assay and analyzed its chemical composition by GCxGC-TOF/MS. The anti-growth activity against MCF-7 and MDA-MB-231 cell lines was assessed by using the ATP viability assay. Cell death mode was investigated with fluorescence staining and ELISA assays. The major components of the flower and trunk were determined as eicosane, heptacosane, 2-propen-1-ol, hexahydrofarnesyl acetone and α-muurolene. HOE caused significant DNA damage at selected doses (250-750 µg/ml) while chromosomal damage was observed at higher concentrations (500 and 750 µg/ml). HOE demonstrated anti-growth activity in a dose-dependent manner between 3.13-100 µg/ml. Pyknotic nuclei were observed at 100 µg/ml concentration of HOE in both cell lines. In conclusion, HOE demonstrated cytotoxic effects in a cell type-dependent manner, however its genotoxic effects were observed at relatively higher doses.

Enhanced cytotoxic activity of doxorubicin through the inhibition of autophagy in triple negative breast cancer cell line.

BACKGROUND: The outcome of triple negative breast cancer is still poor and requires improvement with better therapy options. Autophagy has recently been shown to play a role in anticancer drug resistance. Therefore, we investigated if the effectiveness of doxorubicin was augmented by the inhibition of autophagy. METHODS: MDA-MB-231 was used as a model cell line for triple negative breast cancer and 3-methyladenine was used as an inhibitor of autophagy. Cells were treated with 0.46-1.84µM doxorubicin and 2.5-10µM 3-methyladenine for 48h. Cell death mode was examined with M30 and M65 ELISA assays. ROS level and LDH activity was examined and the cellular acidic compartment of cells was monitored by acridine orange staining. The expression of various autophagy and apoptosis related proteins/genes were evaluated with Western blotting and RT-qPCR respectively. RESULTS: Synergism was observed between the compounds (CI value<1.0). RT-qPCR analysis revealed that the combination resulted in a down-regulation of autophagy-related genes. Moreover, the combination resulted in a different cell death modality, upregulating necroptosis-related genes. This suggests that the mode of cell death may switch from apoptosis to necroptosis, which is a more severe form of cell death, when autophagy is inhibited. These results were further confirmed at protein level by Western blotting. CONCLUSION: Inhibition of autophagy seems to sensitize triple negative breast cancer cells to doxorubicin, warranting further in vivo studies for the proof of this concept. GENERAL SIGNIFICANCE: Autophagy has a key role in drug resistance in MDA-MB-231 cells. Therefore combinatorial approaches may effectively overcome resistance.

Pelargonium quercetorum Agnew induces apoptosis without PARP or cytokeratin 18 cleavage in non-small cell lung cancer cell lines.

Pelargonium species have various uses in folk medicine as traditional remedies, and several of them have been screened for their biological activity, including anticancer. Pelargonium quercetorum Agnew (P. quercetorum) is traditionally used for its anthelminthic activity. However, little is known about its biological activity or its effect on cancer cells. The aim of the present study was to determine the cytotoxic activity of P. quercetorum extract on lung cancer cell lines with varying properties. Following the analyses of its chemical composition, the cytotoxic activity was screened by the adenosine triphosphate viability test. M30-Apoptosense® and M65 EpiDeath® enzyme-linked immunosorbent assays were used to determine the cell death mode (apoptosis vs. necrosis). For apoptosis, additional methods, including Annexin-V-fluorescein isothiocyanate (FITC) and Hoechst 33342 staining, were employed. The cleavage of poly (adenosine diphosphate-ribose) polymerase (PARP) was assayed by western blotting to further dissect the apoptosis mechanism. The methanol extract of P. quercetorum caused cytotoxic activity in a dose-dependent manner. The mode of cell death was apoptosis, as evidenced by the positive staining of the cells for Annexin-V-FITC and the presence of pyknotic nuclei. Notably, neither PARP cleavage nor cytokeratin 18 fragmentation were observed. P.quercetorum caused cell death by an apoptosis mechanism that is slightly different from classical apoptosis. Therefore, future in vivo experiments are required for further understanding of the effect of this plant on cancer cells.

Evaluation of genotoxic and apoptotic potential of Hypericum adenotrichum Spach. in vitro.

Hypericum adenotrichum Spach. is an endemic plant from Turkey that is also used in folk medicine. In this study, following analyses of its chemical composition, the genotoxic/antigenotoxic effects of the methanol extract of H. adenotrichum in human lymphocyte culture were investigated using in vitro sister chromatid exchange, micronucleus and comet assays. In addition, the anti-growth effect of the extract was investigated in human breast cancer cell lines (MCF-7 and MDA-MB-231) using MTT and ATP viability assays. The mode of cell death was determined using fluorescence microscopy and biochemical methods. We found that the H. adenotrichum extract demonstrated cytotoxic and genotoxic effects in a cell type-dependent manner. At selected doses (125-500 µg/ml), the H. adenotrichum extract exhibited significant genotoxic activity in human lymphocytes, whereas it showed anti-growth effects on cancer cell lines between 0.2 and 100 µg/ml concentrations. The mode of cell death in cancer cells was shown to be apoptosis due to the presence of pyknotic nuclei, the cleavage of poly-(ADP-ribose) polymerase (PARP) and/or the activation of caspase-3. These results suggest that H. adenotrichum might show both cytotoxic and genotoxic effects depending on the cell type. This should be taken into account in its use for therapeutic purposes.

Cationic Pd(II)/Pt(II) 5,5-diethylbarbiturate complexes with bis(2-pyridylmethyl)amine and terpyridine: Synthesis, structures,DNA/BSA interactions, intracellular distribution, cytotoxic activity and induction of apoptosis.

Four new cationic Pd(II) and Pt(II) 5,5-diethylbarbiturate (barb) complexes, [M(barb)(bpma)]X·H2O [M = Pd(II), X = Cl (1); M = Pt(II), X = NO3(-) (2)] and [M(barb)(terpy)]NO3·0.5H2O [M = Pd(II) (3); M = Pt(II) (4)], where bpma = bis(2-pyridylmethyl)amine and terpy = terpyridine, were synthesized and characterized by elemental analysis, IR, UV­vis, NMR, ESI-MS and X-ray crystallography. The DNA binding properties of the cationic complexes were investigated by spectroscopic titrations, displacement experiments, viscosity, DNA melting and electrophoresis measurements. The results revealed that the complexes effectively bind to FS-DNA (fish sperm DNA) via intercalative/minor groove binding modes with intrinsic binding constants (Kb) in the range of 0.50 × 10(4)­1.67 × 10(5) M(-1). Absorption, emission and synchronous fluorescence measurements showed strong association of the complexes with protein (BSA) through a static mechanism. The mode of interaction of complexes towards DNA and protein was also supported by molecular docking. Complexes 1 and 3 showed significant nuclear uptake in HT-29 cells. In addition, 1 and 3 showed higher inhibition than cisplatin on the growth of MCF-7 and HT-29 cells and induced apoptosis on these cells much more effectively than the rest of the complexes as evidenced by pyknotic nuclear morphology. The levels of caspase-cleaved cytokeratin 18 (M30 antigen) in HT-29 cells treated with 1 and 3 increased in a dose-dependent manner, suggesting apoptosis. Moreover, qRT-PCR experiments showed that 1 and 3 caused significant increases in the expression of TNFRSF10B in HT-29 cells, indicating the initiation of apoptosis via cell surface death receptors.

Anti-growth effect of a novel trans-dichloridobis[2-(2-hydroxyethyl)pyridine]platinum (II) complex via induction of apoptosis on breast cancer cell lines.

Breast cancer still continues to be the leading cause of cancer-related mortality in women worldwide. Although advances have been made in the treatment of this disease during the past decade, new approaches and novel compounds are urgently needed. The aim of this study was to evaluate the cytotoxic activity of trans-[PtCl2(2-hepy)2] [2-hepy=2-(2-hydroxyethyl) pyridine] on breast cancer cell lines, MCF-7 and MDA-MB-231. The platinum (II) complex was synthesized and characterized by our laboratory working group. Anti-growth effect was assayed by the MTT and ATP viability assays and also monitored real-time using xCELLigence system. The mode of cell death was evaluated by using the fluorescence microscopy (Hoechst 33342+Calcein-AM+Propidium iodide staining), Western blotting (cleaved PARP and caspase 3, total caspase 8), flow cytometry (quantitative analysis of live, early/late apoptotic, dead cells and caspase 3/7 activity) and the RT-PCR (the genes analyzed were BCL-2L10, BIK, BAX, BCL-2, FASLG, HRK, TNFRSF10B, and TNFRSF10A). The platinum (II) complex had anti-growth effect in a dose dependent manner in vitro. Cells were killed by apoptosis as evidenced by the pyknotic nuclei, cleavage of poly-(ADP-ribose) polymerase (PARP) and induction of active caspase-3. These results suggest that the complex might represent a potentially active novel drug for the breast cancer treatment and warrants further studies due to its promising cytotoxic activity.

The M30 assay does not detect apoptosis in epithelial-derived cancer cells expressing low levels of cytokeratin 18.

The primary aim of this study was to compare measurement of apoptosis by M30 immunoreactivity (a biomarker for apoptosis) to other apoptosis assays (morphological assessment of nuclei, Annexin-V-FITC staining, DNA fragmentation and PARP cleavage) in vitro. Caspase-cleaved cytokeratin 18 (M30, ccK18) is only produced in epithelial cells and is regarded as a pharmacodynamic biomarker of apoptotic cell death because it is released from cells during apoptosis induced by chemotherapeutic agents. However, we have observed false negative results using this assay in clinical samples. Therefore, we tested its ability to accurately detect apoptosis in a panel of lung cancer cell lines with a range of clinically approved chemotherapeutic drugs. Three different non-small cell lung cancer (NSCLC) cell lines (A549, H1299, PC3) were used to correlate M30 levels with alternate apoptosis assays. Following successful induction of apoptosis, the A549 cell line showed an increase in M30 levels along with other well-known features of apoptosis, whilst H1299 and PC3 cell lines did not show an increase in M30 levels, even when apoptosis was detected by other means. Further analysis showed that H1299 and PC3 cell lines expressed much lower levels of cytokeratin 18 protein compared to the A549 cell line. Our results suggest that reliable detection of apoptosis via the M30 assay only works when sufficient levels of cytokeratin 18 are present in the cells. This means that the M30 assay may result in false negative results for apoptosis, and as such, the ELISA should be used in conjunction with other assays.

Additive enhancement of apoptosis by TRAIL and fenretinide in metastatic breast cancer cells in vitro.

Successful management of metastatic breast cancer still needs better chemotherapeutic approaches. The combination of fenretinide (4-HPR), a synthetic retinoid inducing apoptosis by ROS generation, and TRAIL, a cell death ligand inducing caspase-dependent apoptosis, might result in more powerful cytotoxic activity. We therefore investigated the cytotoxic activity and resulting cell death mode of this combination in MDA-MB-231 cell line as a representative of metastatic state. Cytotoxicity was assessed by the ATP viability assay while the mode of cell death was determined both morphologically using fluorescence microscopy and biochemically using Western blotting and ELISA. The combination resulted in an additive cytotoxic effect at the doses used. Fragmented and/or pyknotic nuclei, which is a feature of apoptosis, were observed after treatment with fenretinide or TRAIL. However, the combinatorial treatment further increased apoptotic figures. Confirming apoptosis, active caspase-3 and cleaved PARP were increased by fenretinide or TRAIL in both western blotting and ELISA. Again, apoptosis was further increased by the combination. The combination warrants further studies due to its superior cytotoxic activity in the metastatic setting of breast cancer.

Promising anti-growth effects of palladium(II) saccharinate complex of terpyridine by inducing apoptosis on transformed fibroblasts in vitro.

Fibrosarcoma is one of the fatal cancer types and there is still not satisfactory success in its treatment despite new drugs. Therefore, the search for a new compound has been going on. It is currently known that some palladium-based anti-cancer compounds seem to have powerful apoptosis-inducing effects in cancer cells. For this purpose, a palladium(II)-saccharinate complex containing terpyridine which was synthesized by our research group was investigated in terms of its anti-tumor effects against mouse embryonic fibroblast NIH/3T3 (normal cell line) and rat embryonic fibroblast 5RP7 (H-ras transformed cell line) in vitro. The MTT and ATP viability assays were used to determine anti-growth/cytotoxic effects. Cytotoxic activity was confirmed by real time cytotoxicity analysis system. Flow cytometry analysis was further used to determine the mode of cell death (apoptosis/necrosis). Apoptosis was confirmed by triple-staining the cells with Hoechst 33342/PI/Calcein-AM triple and evaluated with fluorescence microscopy. It was found that the compound showed significant anti-growth activity by inducing apoptosis in a dose dependent manner. In conclusion, taking into account the cytotoxic activity of the compound at even relatively lower doses, in vivo experiments to elucidate its potential use for the treatment of fibrosarcoma are warranted.

Palladium(II) saccharinate complexes with bis(2-pyridylmethyl)amine induce cell death by apoptosis in human breast cancer cells in vitro.

The outcomes of breast cancer patients are still poor although new compounds have recently been introduced into the clinic. Therefore, novel chemical approaches are required. In the present study, palladium(II) and corresponding platinum(II) complexes containing bis(2-pyridylmethyl)amine (bpma) and saccharine were synthesized and tested against human breast cancer cell lines, MCF-7 and MDA-MB-231, in vitro. Cytotoxicity was first screened by the MTT assay and the results were further confirmed by the ATP assay. The palladium complexes 1 and 3 yielded stronger cytotoxicity than the corresponding platinum complexes 2 and 4 at the same doses. The palladium complex 3 was found to be the most cytotoxic one. Therefore, a more comprehensive study was carried out with this complex only. The mode of cell death was determined morphologically under fluorescent microscope and biochemically with detection of active caspase-3 and PARP cleavage by Western blot. Changes in apoptosis-related gene expressions were measured with qPCR. It was demonstrated that complex 3 caused cell death by apoptosis determined by fluorescence imaging and Western blot. As a sign of apoptosis, PARP was cleaved in both of the cell lines. In addition, caspase-3 was cleaved in MDA-MB-231 cells while this cleavage was not observed in MCF-7. The results show that the complex 3 is a promising anti-cancer compound against breast cancer with an IC50 value of 3.9 µM for MCF-7 and 4.2 µM for MDA-MB-231 cells, which warrants further animal experiments.

Combination of fenretinide and indole-3-carbinol results in synergistic cytotoxic activity inducing apoptosis against human breast cancer cells in vitro.

The outcome in patients with breast cancer is not satisfactory to date, although new chemotherapy regimens have been introduced in clinics. Therefore, novel approaches are required for better management of patients with breast cancer. In this study, we tested the cytotoxic activity of a new combination of fenretinide, a synthetic retinoid, with indole-3-carbinol, a natural product present in vegetables such as broccoli and cabbage, against MCF-7 (estrogen receptor-positive) and MDA-MB-231 (estrogen receptor-negative) cell lines. It has been found that the combination resulted in more powerful cytotoxic activity, by induction of apoptosis, compared with that when they were used singly. In conclusion, this novel combination warrants in-vivo experiments to elucidate its possible use in the treatment of breast cancer.

Cell death-inducing effect of novel palladium(II) and platinum(II) complexes on non-small cell lung cancer cells in vitro.

PURPOSE: Treatment for lung cancer is still far from satisfying rates. Therefore, there is a need for novel anticancer agents. For this purpose, novel platinum and palladium complexes {[Pd(sac)(terpy)](sac)·4H(2)O (Complex 1), [Pt(sac)(terpy)](sac)·5H(2)O (Complex 2), [PdCl(terpy)](sac)·2H(2)O (Complex 3), [PtCl(terpy)](sac)·2H(2)O (Complex 4)} have been tested against three different non-small cell lung cancer cell lines (A549, H1299, PC-3). METHODS: Growth-inhibiting effects have been tested by the MTT and ATP viability assays. Apoptosis has been detected by the caspase-cleaved cytokeratin 18 (M30-antigen) assay. Necrosis has been detected by staining the cells with fluorescent dyes. Mitotic index has been calculated by counting the mitotic figures after staining with hematoxylin. RESULTS: The complex 3 exhibited significant anti-growth effects, and its anti-growth effect was more powerful than that of cisplatin that is a standard chemotherapeutic agent for this type of cancer. The complexes did not induce apoptosis, while necrosis clearly took place. CONCLUSIONS: Novel Pd(II) complex ([PdCl(terpy)](sac)·2H(2)O) seems to represent a potentially active drug against non-small cell lung cancer cell lines, and further studies in vivo are warranted.