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This study was focused on investigating the antiproliferative effects of chalcone hybrids in melanoma cancer cells. Among seven chalcone hybrids, the chalcone-acridine hybrid 1C was the most potent and was selected for further antiproliferative mechanism studies. This in vitro study revealed the potent antiproliferative effect of 1C via cell cycle arrest and apoptosis induction. Cell cycle arrest at the G2/M phase was associated with modulation of expression or phosphorylation of specific cell cycle-associated proteins (cyclin B1, p21, and ChK1), tubulins, as well as with the activation of the DNA damage response pathway. Chalcone 1C also induced apoptosis accompanied by mitochondrial dysfunction evidenced by a decrease in mitochondrial membrane potential, increase in Bax/Bcl-xL ratio and cytochrome c release followed by caspase 3/7 activation. In addition, increased phosphorylation of MAP kinases (Erk1/2, p38 and JNK) was observed in chalcone 1C-treated melanoma cells. The strong antiproliferative activities of this chalcone-acridine hybrid suggest that it may be useful as an antimelanoma agent in humans.

Simple Summary Despite the important progress in cancer treatment in the past decades, the mortality rates in some types of cancer have not significantly decreased. Therefore, the search for novel anticancer drugs has become a topic of great interest. Chalcones, precursors of flavonoid synthesis in plants, have been documented as natural compounds with pleiotropic biological effects including antiproliferative/anticancer activity. This article focuses on the knowledge on molecular mechanisms of antiproliferative action of chalcones and draws attention to this group of natural compounds that may be of importance in the treatment of cancer disease. Abstract Although great progress has been made in the treatment of cancer, the search for new promising molecules with antitumor activity is still one of the greatest challenges in the fight against cancer due to the increasing number of new cases each year. Chalcones (1,3-diphenyl-2-propen-1-one), the precursors of flavonoid synthesis in higher plants, possess a wide spectrum of biological activities including antimicrobial, anti-inflammatory, antioxidant, and anticancer. A plethora of molecular mechanisms of action have been documented, including induction of apoptosis, autophagy, or other types of cell death, cell cycle changes, and modulation of several signaling pathways associated with cell survival or death. In addition, blockade of several steps of angiogenesis and proteasome inhibition has also been documented. This review summarizes the basic molecular mechanisms related to the antiproliferative effects of chalcones, focusing on research articles from the years January 2015–February 2021.

Background/aim A series of experiments on HeLa cells were conducted to provide new information concerning the anti-cancer properties of jaspine B hydrochloride (JBH). Materials and methods HeLa cells treated with 0.5 μmol/l JBH for 24, 48, and 72 h underwent flow cytometric analysis of the cell cycle, and measurement of phosphatidylserine externalization, mitochondrial membrane potential (MMP), casp-3 activation, cleavage of PARP, ceramide levels, aSMase activity, and Bcl-2 release. nSMase activity was measured by a colorimetric assay. Gene expression was determined by qRT-PCR. Immunocytochemistry was performed to detect p21 and p27 expression. Results JBH-induced apoptosis in HeLa cells associated with externalization of phosphatidylserine, reduced MMP, activation of casp-3, and cleavage of PARP as well as up-regulation of TNF-α, FasL, and casp-8. Significant increase in nSMase activity, ceramide levels, Bcl-2 release (predominantly in the inactive form), and pro-apoptotic nuclear localization of p21 and p27 were also detected. Conclusion JBH-induced apoptosis in HeLa cells is associated with disrupted sphingolipid homeostasis resulting in increased ceramide levels.

Over the past decades, natural products have emerged as promising agents with multiple biological activities. Many studies suggest the antioxidant, antiangiogenic, antiproliferative and anticancer effects of chalcones and their derivatives. Based on these findings, we decided to evaluate the effects of the newly synthetized chalcone L1 in a human cervical carcinoma cell (HeLa) model. Presented results were obtained by western blot and flow cytometric analyses, live cell imaging and antimigratory potential of L1 in HeLa cells was demonstrated by scratch assay. In the present study, we proved the role of L1 as an effective agent with antiproliferative activity supported by G2/M cell cycle arrest and apoptosis. Moreover, we proved that L1 is involved in modulating Transforming Growth Factor-β1 (TGF-β) signal transduction through Smad proteins and it also modulates other signalling pathways including Akt, JNK, p38 MAPK, and Erk1/2. The involvement of L1 in epithelial-to-mesenchymal transition was demonstrated by the regulation of N-cadherin, E-cadherin, and MMP-9 levels. Here, we also evaluated the effect of conditioned medium from BJ-5ta human foreskin fibroblasts in HeLa cell cultures with subsequent L1 treatment. Taken together, these data suggest the potential role of newly synthesized chalcone L1 as an anticancer-tumour microenvironment modulating agent.

Comprehensive scientific data provide evidence that isolated phytochemicals or whole plant foods may beneficially modify carcinogenesis. The aim of this study was to evaluate the oncostatic activities of Rhus coriaria L. (sumac) using animal models (rat and mouse), and cell lines of breast carcinoma. R. coriaria (as a powder) was administered through the diet at two concentrations (low dose: 0.1% (w/w) and high dose: 1 % (w/w)) for the duration of the experiment in a syngeneic 4T1 mouse and chemically-induced rat mammary carcinoma models. After autopsy, histopathological and molecular analyses of tumor samples in rodents were performed. Moreover, in vitro analyses using MCF-7 and MDA-MB-231 cells were conducted. The dominant metabolites present in tested R. coriaria methanolic extract were glycosides of gallic acid (possible gallotannins). In the mouse model, R. coriaria at a higher dose (1%) significantly decreased tumor volume by 27% when compared to controls. In addition, treated tumors showed significant dose-dependent decrease in mitotic activity index by 36.5% and 51% in comparison with the control group. In the chemoprevention study using rats, R. coriaria at a higher dose significantly reduced the tumor incidence by 20% and in lower dose non-significantly reduced tumor frequency by 29% when compared to controls. Evaluations of the mechanism of oncostatic action using valid clinical markers demonstrated several positive alterations in rat tumor cells after the treatment with R. coriaria. In this regard, histopathological analysis of treated tumor specimens showed robust dose-dependent decrease in the ratio of high-/low-grade carcinomas by 66% and 73% compared to controls. In treated rat carcinomas, we found significant caspase-3, Bax, and Bax/Bcl-2 expression increases, on the other side, a significant down-regulation of Bcl-2, Ki67, CD24, ALDH1, and EpCam expressions and MDA levels. When compared to control specimens, evaluation of epigenetic alterations in rat tumor cells in vivo showed significant dose-dependent decrease in lysine methylation status of H3K4m3 and H3K9m3 and dose-dependent increase in lysine acetylation in H4K16ac levels (H4K20m3 was not changed) in treated groups. However, only in lower dose of sumac were significant decreases in the expression of oncogenic miR210 and increase of tumor-suppressive miR145 (miR21, miR22, and miR155 were not changed) observed. Finally, only in lower sumac dose, significant decreases in methylation status of three out of five gene promoters&ndash, ATM, PTEN, and TIMP3 (PITX2 and RASSF1 promoters were not changed). In vitro evaluations using methanolic extract of R. coriaria showed significant anticancer efficacy in MCF-7 and MDA-MB-231 cells (using Resazurin, cell cycle, annexin V/PI, caspase-3/7, Bcl-2, PARP, and mitochondrial membrane potential analyses). In conclusion, sumac demonstrated significant oncostatic activities in rodent models of breast carcinoma that were validated by mechanistic studies in vivo and in vitro.

Chalcones are naturally occurring phytochemicals with diverse biological activities including antioxidant, antiproliferative, and anticancer effects. Some studies indicate that the antiproliferative effect of chalcones may be associated with their pro-oxidant effect. In the present study, we evaluated contribution of oxidative stress in the antiproliferative effect of acridine chalcone 1C ((2 E)-3-(acridin-9-yl)-1-(2,6-dimethoxyphenyl)prop-2-en-1-one) in human colorectal HCT116 cells. We demonstrated that chalcone 1C induced oxidative stress via increased reactive oxygen/nitrogen species (ROS/RNS) and superoxide production with a simultaneous weak adaptive activation of the cellular antioxidant defence mechanism. Furthermore, we also showed chalcone-induced mitochondrial dysfunction, DNA damage, and apoptosis induction. Moreover, activation of mitogen activated phosphokinase (MAPK) signalling pathway in 1C-treated cancer cells was also observed. On the other hand, co-treatment of cells with strong antioxidant, N-acetyl cysteine (NAC), significantly attenuated all of the above-mentioned effects of chalcone 1C, that is, decreased oxidant production, prevent mitochondrial dysfunction, DNA damage, and induction of apoptosis, as well as partially preventing the activation of MAPK signalling. Taken together, we documented the role of ROS in the antiproliferative/pro-apoptotic effects of acridine chalcone 1C. Moreover, these data suggest that this chalcone may be useful as a promising anti-cancer agent for treating colon cancer.

It is supposed that plant functional foods, rich in phytochemicals, may potentially have preventive effects in carcinogenesis. In this study, the anticancer effects of cloves in the in vivo and in vitro mammary carcinoma model were assessed. Dried flower buds of cloves (CLOs) were used at two concentrations of 0.1% and 1% through diet during 13 weeks after the application of chemocarcinogen. After autopsy, histopathological and immunohistochemical analyses of rat mammary carcinomas were performed. Moreover, in vitro evaluation using MCF‐7 cells was carried out. Dietary administered CLO caused the dose‐dependent decrease in tumour frequency by 47.5% and 58.5% when compared to control. Analysis of carcinoma cells in animals showed bcl‐2, Ki67, VEGFA, CD24 and CD44 expression decrease and Bax, caspase‐3 and ALDH1 expression increase after high‐dose CLO administration. MDA levels were substantially decreased in rat carcinomas in both CLO groups. The evaluation of histone modifications revealed increase in lysine trimethylations and acetylations (H4K20me3, H4K16ac) in carcinomas after CLO administration. TIMP3 promoter methylation levels of CpG3, CpG4, CpG5 islands were altered in treated cancer cells. An increase in total RASSF1A promoter methylation (three CpG sites) in CLO 1 group was found. In vitro studies showed antiproliferative and pro‐apoptotic effects of CLO extract in MCF‐7 cells (analyses of cytotoxicity, Brdu, cell cycle, annexin V/PI, caspase‐7, Bcl‐2 and mitochondrial membrane potential). This study showed a significant anticancer effect of clove buds in the mammary carcinoma model in vivo and in vitro.

Naturally-occurring mixtures of phytochemicals present in plant foods are proposed to possess tumor-suppressive activities. In this work, we aimed to evaluate the antitumor effects of Thymus vulgaris L. in in vivo and in vitro mammary carcinoma models. Dried T. vulgaris (as haulm) was continuously administered at two concentrations of 0.1% and 1% in the diet in a chemically-induced rat mammary carcinomas model and a syngeneic 4T1 mouse model. After autopsy, histopathological and molecular analyses of rodent mammary carcinomas were performed. In addition, in vitro evaluations using MCF-7 and MDA-MB-231 cells were carried out. In mice, T. vulgaris at both doses reduced the volume of 4T1 tumors by 85% (0.1%) and 84% (1%) compared to the control, respectively. Moreover, treated tumors showed a substantial decrease in necrosis/tumor area ratio and mitotic activity index. In the rat model, T. vulgaris (1%) decreased the tumor frequency by 53% compared to the control. Analysis of the mechanisms of anticancer action included well-described and validated diagnostic and prognostic markers that are used in both clinical approach and preclinical research. In this regard, the analyses of treated rat carcinoma cells showed a CD44 and ALDH1A1 expression decrease and Bax expression increase. Malondialdehyde (MDA) levels and VEGFR-2 expression were decreased in rat carcinomas in both the T. vulgaris treated groups. Regarding the evaluations of epigenetic changes in rat tumors, we found a decrease in the lysine methylation status of H3K4me3 in both treated groups (H3K9m3, H4K20m3, and H4K16ac were not changed), up-regulations of miR22, miR34a, and miR210 expressions (only at higher doses), and significant reductions in the methylation status of four gene promoters&mdash, ATM serin/threonine kinase, also known as the NPAT gene (ATM), Ras-association domain family 1, isoform A (RASSF1), phosphatase and tensin homolog (PTEN), and tissue inhibitor of metalloproteinase-3 (TIMP3) (the paired-like homeodomain transcription factor (PITX2) promoter was not changed). In vitro study revealed the antiproliferative and proapoptotic effects of essential oils of T. vulgaris in MCF-7 and MDA-MB-231 cells (analyses of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS), 5-bromo-20-deoxyuridine (BrdU), cell cycle, annexin V/PI, caspase-3/7, Bcl-2, PARP, and mitochondrial membrane potential). T. vulgaris L. demonstrated significant chemopreventive and therapeutic activities against experimental breast carcinoma.

Naturally-occurring mixtures of phytochemicals present in plant foods are proposed to possess tumor-suppressive activities. In this work, we aimed to evaluate the antitumor effects of Thymus vulgaris L. in in vivo and in vitro mammary carcinoma models. Dried T. vulgaris (as haulm) was continuously administered at two concentrations of 0.1% and 1% in the diet in a chemically-induced rat mammary carcinomas model and a syngeneic 4T1 mouse model. After autopsy, histopathological and molecular analyses of rodent mammary carcinomas were performed. In addition, in vitro evaluations using MCF-7 and MDA-MB-231 cells were carried out. In mice, T. vulgaris at both doses reduced the volume of 4T1 tumors by 85% (0.1%) and 84% (1%) compared to the control, respectively. Moreover, treated tumors showed a substantial decrease in necrosis/tumor area ratio and mitotic activity index. In the rat model, T. vulgaris (1%) decreased the tumor frequency by 53% compared to the control. Analysis of the mechanisms of anticancer action included well-described and validated diagnostic and prognostic markers that are used in both clinical approach and preclinical research. In this regard, the analyses of treated rat carcinoma cells showed a CD44 and ALDH1A1 expression decrease and Bax expression increase. Malondialdehyde (MDA) levels and VEGFR-2 expression were decreased in rat carcinomas in both the T. vulgaris treated groups. Regarding the evaluations of epigenetic changes in rat tumors, we found a decrease in the lysine methylation status of H3K4me3 in both treated groups (H3K9m3, H4K20m3, and H4K16ac were not changed); up-regulations of miR22, miR34a, and miR210 expressions (only at higher doses); and significant reductions in the methylation status of four gene promoters—ATM serin/threonine kinase, also known as the NPAT gene (ATM); Ras-association domain family 1, isoform A (RASSF1); phosphatase and tensin homolog (PTEN); and tissue inhibitor of metalloproteinase-3 (TIMP3) (the paired-like homeodomain transcription factor (PITX2) promoter was not changed). In vitro study revealed the antiproliferative and proapoptotic effects of essential oils of T. vulgaris in MCF-7 and MDA-MB-231 cells (analyses of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS); 5-bromo-20-deoxyuridine (BrdU); cell cycle; annexin V/PI; caspase-3/7; Bcl-2; PARP; and mitochondrial membrane potential). T. vulgaris L. demonstrated significant chemopreventive and therapeutic activities against experimental breast carcinoma.

In the present study, we investigated the antiproliferative activity of Naja ashei full venom (NAV) on human colorectal cancer cells. The NAV-induced antiproliferative effect was associated with cell cycle arrest in S phase and increased number of cells with sub G0/G1 DNA content, which is considered a marker of apoptosis. Apoptosis has also been confirmed with annexin V/PI staining. Furthermore, flow cytometric analysis revealed loss of mitochondrial membrane potential with concomitant increase in cytochrome c and Smac/DIABLO protein content. These effects were associated with the activation of caspase-9 and caspase-3, as well as with PARP cleavage. Moreover, phosphorylation of antiapoptotic Bcl-2 protein in NAV-treated HCT116 was observed. In conclusion, our study for the first time documented antiproliferative/pro-apoptotic effect of NAV in colorectal cancer cells. Our results strongly suggest the involvement of mitochondria in NAV induced apoptosis of cancer cells. Future studies are needed to further examine the potential of NAV in the treatment of colon cancer.

Background Lichens produce a huge diversity of bioactive compounds with several biological effects. Gyrophoric acid (GA) is found in high concentrations in the common lichen Umbilicaria hirsuta, however evidence for biological activity was limited to anti-proliferative activity described on several cancer cell lines. Methods We developed and validated a new protocol for GA isolation, resulting in a high yield of highly pure GA (validated by HPLC and NMR) in an easy and time saving manner. Anti-proliferative and pro-apoptotic activity, oxygen radicals formation and stress/survival proteins activity changes was study by flow cytometry. Results The highly purified GA showed anti-proliferative activity against HeLa (human cervix carcinoma) and other tumor cells. Moreover, GA threated cells showed a significant increase in caspase-3 activation followed by PARP cleavage, PS externalization and cell cycle changes mediated by oxidative stress. Production of oxygen radicals led to DNA damage and changes in stress/survival pathways activation. Conclusions GA treatment on HeLa cells clearly indicates ROS production and apoptosis as form of occurred cell death. Moreover, DNA damage and changing activity of stress/survival proteins as p38MAPK, Erk1/2 and Akt mediated by GA treatment confirm pro-apoptotic potential. The pharmacological potential of U. hirsuta derived GA is discussed. Electronic supplementary material The online version of this article (10.1186/s12906-019-2631-4) contains supplementary material, which is available to authorized users.

The effect of dietary administered young barley containing a mixture of phytochemicals to female rats for the prevention of N-methyl-N-nitrosourea-induced mammary carcinogenesis was evaluated. After carcinogen administration (14 wk), mammary tumors were removed and prepared for histopathological and immunohistochemical analysis. Moreover, in vitro evaluation of possible mechanisms in MCF-7 breast cancer cell line was performed. Barley (0.3%) demonstrated mild antitumor effect in mammary carcinogenesis, yet 3% barley did not further improve this effect. Immunohistochemical analysis of rat tumor cells in treated groups showed significant increase in caspase-3 expression and significant reduction in Ki67 expression. In addition, 3% barley significantly decreased dityrosine levels versus control. Barley in higher dose significantly decreased serum low-density lipoprotein-cholesterol in rats. In vitro studies showed that barley significantly decreased survival of MCF-7 cells in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and significantly decreased 5-bromo-20-deoxyuridine incorporation versus control. Barley prevented cell cycle progression and extended incubation with barley showed significant increase in the percentage of annexin V/propidium iodide-positive MCF-7 cells. Our results propose an antitumor effect for the mixture of phytochemicals present in young barley in a breast cancer model.

Comprehensive oncology research suggests an important role of phytochemicals or whole plant foods in the modulation of signaling pathways associated with anticancer action. The goal of this study is to assess the anticancer activities of Cinnamomum zeylanicum L. using rat, mouse, and cell line breast carcinoma models. C. zeylanicum (as bark powder) was administered in the diet at two concentrations of 0.1% (w/w) and 1% (w/w) during the whole experiment in chemically induced rat mammary carcinomas and a syngeneic 4T1 mouse model. After autopsy, histopathological and molecular evaluations of mammary gland tumors in rodents were carried out. Moreover, in vitro analyses using MCF-7 and MDA-MB-231 cells were performed. The dominant metabolites present in the tested C. zeylanicum essential oil (with relative content over 1%) were cinnamaldehyde, cinnamaldehyde dimethyl acetal, cinnamyl acetate, eugenol, linalool, eucalyptol, limonene, o-cymol, and &alpha, terpineol. The natural mixture of mentioned molecules demonstrated significant anticancer effects in our study. In the mouse model, C. zeylanicum at a higher dose (1%) significantly decreased tumor volume by 44% when compared to controls. In addition, treated tumors showed a significant dose-dependent decrease in mitotic activity index by 29% (0.1%) and 45.5% (1%) in comparison with the control group. In rats, C. zeylanicum in both doses significantly reduced the tumor incidence by 15.5% and non-significantly suppressed tumor frequency by more than 30% when compared to controls. An evaluation of the mechanism of anticancer action using valid oncological markers showed several positive changes after treatment with C. zeylanicum. Histopathological analysis of treated rat tumor specimens showed a significant decrease in the ratio of high-/low-grade carcinomas compared to controls. In treated rat carcinomas, we found caspase-3 and Bax expression increase. On the other hand, we observed a decrease in Bcl-2, Ki67, VEGF, and CD24 expressions and MDA levels. Assessment of epigenetic changes in rat tumor cells in vivo showed a significant decrease in lysine methylation status of H3K4m3 and H3K9m3 in the high-dose treated group, a dose-dependent increase in H4K16ac levels (H4K20m3 was not changed), down-regulations of miR21 and miR155 in low-dose cinnamon groups (miR22 and miR34a were not modulated), and significant reduction of the methylation status of two out of five gene promoters&mdash, ATM and TIMP3 (PITX2, RASSF1, PTEN promoters were not changed). In vitro study confirmed results of animal studies, in that the essential oil of C. zeylanicum displayed significant anticancer efficacy in MCF-7 and MDA-MB-231 cells (using MTS, BrdU, cell cycle, annexin V/PI, caspase-3/7, Bcl-2, PARP, and mitochondrial membrane potential analyses). As a conclusion, C. zeylanicum L. showed chemopreventive and therapeutic activities in animal breast carcinoma models that were also significantly confirmed by mechanistic evaluations in vitro and in vivo.

Cancerous tissue transformation developing usually over years or even decades of life is a highly complex process involving strong stressors damaging DNA, chronic inflammation, comprehensive interaction between relevant molecular pathways, and cellular cross-talk within the neighboring tissues. Only the minor part of all cancer cases are caused by inborn predisposition; the absolute majority carries a sporadic character based on modifiable risk factors which play a central role in cancer prevention. Amongst most promising candidates for dietary supplements are bioactive phytochemicals demonstrating strong anticancer effects. Abundant evidence has been collected for beneficial effects of flavonoids, carotenoids, phenolic acids, and organosulfur compounds affecting a number of cancer-related pathways. Phytochemicals may positively affect processes of cell signaling, cell cycle regulation, oxidative stress response, and inflammation. They can modulate non-coding RNAs, upregulate tumor suppressive miRNAs, and downregulate oncogenic miRNAs that synergically inhibits cancer cell growth and cancer stem cell self-renewal. Potential clinical utility of the phytochemicals is discussed providing examples for chemoprevention against and therapy for human breast cancer. Expert recommendations are provided in the context of preventive medicine.

In the present study, we investigated antiproliferative activity of seven newly synthesized chalcone derivatives. Among tested compounds, (2 E)-3-(acridin-9-yl)-1-(2,6-dimethoxyphenyl)prop-2-en-1-one (1C) was the most potent with IC50 = 4.1 μmol/L in human colorectal HCT116 cells and was selected for further studies. Inhibition of cell proliferation was associated with cell cycle arrest in G2/M phase and dysregulation of α, α1 and β5 tubulins. Moreover, 1C caused disruption of the mitochondrial membrane potential and increased number of cells with sub G0/G1 DNA content which is considered as marker of apoptosis. Apoptosis was confirmed by annexin V/PI and AO/PI staining. Furthermore, we found increased concentration of cytochrome c, Smac/Diablo and increased caspase-3 and caspase-9 activity, cleavage of PARP as well as activation of DNA repair mechanisms in 1C-treated HCT116 cancer cells. Moreover this chalcone derivative up-regulated proapoptotic Bax expression and down-regulated antiapoptotic Bcl-2 and Bcl-xL expression. Additionally, 1C treatment led to modulation of MAPKs and Akt signalling pathways. In conclusion, our data showed ability of 1C to suppress cancel cell growth and provide the rationale for further in vivo study.

Fruit and vegetable intake is inversely correlated with cancer; thus, it is proposed that an extract of phytochemicals as present in whole fruits, vegetables, or grains may have anti-carcinogenic properties. Thus, the anti-tumour effects of fruit peel polyphenols (Flavin7) in the chemoprevention of N-methyl-N-nitrosourea-induced mammary carcinogenesis in female rats were evaluated.Lyophilized substance of Flavin7 (F7) was administered at two concentrations of 0.3 and 3 % through diet. The experiment was terminated 14 weeks after carcinogen administration, and mammary tumours were removed and prepared for histopathological and immunohistochemical analysis. In addition, using an in vitro cytotoxicity assay, apoptosis and proliferation after F7 treatment in human breast adenocarcinoma (MCF-7) cells were performed.High-dose F7 suppressed tumour frequency by 58 % (P0.001), tumour incidence by 24 % (P0.05), and lengthened latency by 8 days (P0.05) in comparison with the control rats, whereas lower dose of F7 was less effective. Histopathological analysis of tumours showed significant decrease in the ratio of high-/low-grade carcinomas after high-dose F7 treatment. Immunohistochemical analysis of rat carcinoma cells in vivo found a significant increase in caspase-3 expression and significant decrease in Bcl-2, Ki67, and VEGFR-2 expression in the high-dose group. Both doses demonstrated significant positive effects on plasma lipid metabolism in rats. F7 significantly decreased survival of MCF-7 cells in vitro in MTT assay by dose- and time-dependent manner compared to control. F7 prevented cell cycle progression by significant enrichment in G1 cell populations. Incubation with F7 showed significant increase in the percentage of annexin V-/PI-positive MCF-7 cells and DNA fragmentation.Our results reveal a substantial tumour-suppressive effect of F7 in the breast cancer model. We propose that the effects of phytochemicals present in this fruit extract are responsible for observed potent anti-cancer activities.

Polyphenols represent a large group of natural substances with different biological properties. Currently, polyphenols are well studied due to their free radicals' scavenging and antioxidant activities. However, some studies indicate that polyphenols also exhibit pro-oxidant properties. In this study, the possible involvement of the pro-oxidant activities of fruit polyphenols was investigated in relation to apoptosis induction. To determine the type of cell death induced by fruit polyphenols (Flavine; F7), we assessed a series of assays, including measurements of caspase-7 activation, membrane mitochondrial potential changes, reactive oxygen (ROS) and nitrogen species production, lipid peroxidation, antioxidant enzymes activities, and PARP cleavage. Moreover, the effect of F7 on selected pro- and antisurvival signaling pathways was determined. We demonstrated that fruit polyphenols induced caspase-dependent cell death associated with increased oxidative stress. We also showed fruit polyphenol-mediated release of mitochondrial pro- and antiapoptotic proteins of the Bcl-2 family and modulation activity of the Akt, p38 MAPK, and Erk 1/2 pathways as well as the signaling of ROS-mediated DNA damage. Our data demonstrated that fruit peel polyphenols suppressed breast cancer cell growth through increased intracellular oxidative stress and the activation of p38 MAPK and de-activation of the Erk 1/2 and Akt signaling pathways.

This study was designed to examine the in vitro antiproliferative effect of brassinin and its derivatives on human cancer cell lines. Among seven tested compounds, homobrassinin (K1; N-[2-(indol-3-yl)ethyl]-S-methyldithiocarbamate) exhibited the most potent activity with IC50 = 8.0 μM in human colorectal Caco2 cells and was selected for further studies. The flow cytometric analysis revealed a K1-induced increase in the G2/M phase associated with dysregulation of α-tubulin, α1-tubulin and β5-tubulin expression. These findings suggest that the inhibitory effect of K1 can be mediated via inhibition of microtubule formation. Furthermore, simultaneously with G2/M arrest, K1 also increased population of cells with sub-G1 DNA content which is considered to be a marker of apoptotic cell death. Apoptosis was also confirmed by annexin V/PI double staining, DNA fragmentation assay and chromatin condensation assay. The apoptosis was associated with the loss of mitochondrial membrane potential (MMP), caspase-3 activation as well as intracellular reactive oxygen species (ROS) production. Moreover, the antioxidant Trolox blocked ROS production, changes in MMP and decreased K1 cytotoxicity, which confirmed the important role of ROS in cell apoptosis. Taken together, our data demonstrate that K1 induces ROS-dependent apoptosis in Caco2 cells and provide the rationale for further in vivo anticancer investigation.

Photoactivated hypericin increased production of reactive oxygen species in human breast adenocarcinoma MCF-7 as well as in MDA-MB-231 cells 1h after photodynamic therapy. On the other hand, reactive oxygen species dropped 3h after photodynamic therapy with hypericin, but only in MCF-7 cells, whereas in MDA-MB-231 cells remained elevated. The difference in the dynamics of reactive oxygen species after hypericin activation was related to increased activity of SOD-2 in MCF-7 cells compared to MDA-MB-231 cells. Indeed, photodynamic therapy with hypericin significantly increased SOD-2 activity in MCF-7 cells, but only slightly in MDA-MB-231 cells. In this regard, SOD-2 activity correlated well with enhanced both mRNA expression as well as SOD-2 protein level in MCF-7 cells. The role of SOD-2 in the resistance of MCF-7 cells to photodynamic therapy with hypericin was monitored using SOD-2 inhibitor - 2-methoxyestradiol. Interestingly, the combination of photodynamic therapy with hypericin and methoxyestradiol sensitized MCF-7 cells to photodynamic therapy and significantly reduced its clonogenic ability. Furthermore, methoxyestradiol potentiated the activation of caspase 3/7 and apoptosis induced by photodynamic therapy with hypericin.

This study examined the antiproliferative effects of β-escin (E) in cancer cells. The study showed that E inhibited cancer cells growth in a dose-dependent manner. The flow cytometric analysis revealed an escin-induced increase in the sub-G1 DNA content, which is considered to be a marker of apoptosis. Apoptosis was also confirmed by annexin V staining and DNA fragmentation assay. These effects were associated with increased generation of reactive oxygen species (ROS), caspase-3 activation and decreased mitochondrial membrane potential (MMP). Moreover, escin decreased mitochondrial protein content and mitochondrial fluorescence intensity as well as caused depletion of glutathione (GSH). However, activity of glutathione peroxidase (GPx) and glutathione reductase (GR) was not significantly changed in escin-treated cells. In conclusion, our results demonstrated that E has apoptotic effects in human cancer cells through the mechanisms involving mitochondrial perturbation. Although the exact mechanism needs to be investigated further, it can be concluded that E may be a useful candidate agent for cancer treatment.

Several photodynamically-active substances and farnesyltransferase inhibitors are currently being investigated as promising anticancer drugs. In this study, the combined effect of hypericin (the photodynamically-active pigment from Hypericum perforatum) and selective farnesyltransferase inhibitor manumycin (manumycin A; the selective farnesyltransferase inhibitor from Streptomyces parvulus) on HT-29 adenocarcinoma cells was examined. We found that the combination treatment of cells with photoactivated hypericin and manumycin resulted in enhanced antiproliferative and apoptotic response compared to the effect of single treatments. This was associated with increased suppression of clonogenic growth, S phase cell cycle arrest, elevated caspase-3/7 activity and time-dependent total cleavage of procaspase-3 and lamin B, cleavage of p21Bax into p18Bax and massive PARP cleavage. Moreover, we found that the apoptosis-inducing factor is implicated in signaling events triggered by photoactivated hypericin. Our results showed the relocalization of apoptosis-inducing factor (AIF) to the nuclei after hypericin treatment. In addition, we discovered that not only manumycin but also photoactivated hypericin induced the reduction of total Ras protein level. Manumycin decreased the amount of farnesylated Ras, and the combination treatment decreased the amount of both farnesylated and non-farnesylated Ras protein more dramatically. The present findings indicate that the inhibition of Ras processing may be the determining factor for enhancing the antiproliferative and apoptotic effects of combination treatment on HT-29 cells.

Since many studies have suggested the impact of dietary polyunsaturated fatty acids on cancer progression and prognosis, there is an assumption of possible pre-sensitizing effects of their application in combined treatment. The present work evaluates modulation of photodynamic therapy (PDT) with hypericin by pre-treatment with n-3 and n-6 fatty acids in HT-29 and HeLa tumour cells. We observed stimulation of cytotoxic effects by docosahexaenoic acid (n-3) and arachidonic acid (n-6) in several stages of action in both cell lines. Treatment with either fatty acids or photodynamic therapy alone induced apoptosis in a dose- and time-dependent manner; however the effect was even more striking in mutual combination applied as pre-treatment with fatty acids prior to photodynamic therapy. Moreover, the combination also induced changes in membrane lipid composition leading to alteration in cell membrane fluidity. Increased toxicity of combined treatment was also confirmed by the presence of oxidative stress demonstrated by ROS production, RNS accumulation and increased presence of lipoperoxides. In conclusion, we suggest that pre-treatment with polyunsaturated fatty acids may contribute to cytotoxic effects induced by photodynamic therapy with hypericin.

Photodynamic therapy (PDT) is a flexible multi-target therapeutic approach. One of the main requirements of successful PDT is sufficient intracellular concentration of an applicable photosensitizer. Mechanisms of anticancer drug elimination by tumour cells are mostly linked to the elevated expression and activity of P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1), breast cancer resistance protein (BCRP) and P450 monooxygenases. The interaction of hypericin with this cell drug-defence system is still unclear. We report here for the first time increased activity of MRP1 and BCRP in HT-29 colon cancer cells treated with hypericin per se. On the contrary, pre-treatment with proadifen (SKF525A) affected the function of MRP1 and BCRP leading to increased hypericin content, which might indicate a possible link between proadifen and these ABC transporter proteins. Subsequent enhanced intracellular oxidative stress was accompanied by loss of mitochondrial membrane potential, activation of caspase-9 and -3, PARP cleavage and onset of apoptosis. In conclusion, our study suggests that drug efflux transporters MRP1 and BCRP affect the pharmacokinetics of hypericin in HT-29 colon adenocarcinoma cells, and the action of hypericin-mediated PDT (HY-PDT) should be modulated by pre-treatment with their specific inhibitors.

There has been a considerable interest in the identification of natural plant foods for developing effective agents against cancer. Thus, the anti-tumour effects of oregano in the in vivo and in vitro breast cancer model were evaluated. Lyophilized oregano (ORE) was administered at two concentrations of 0.3 and 3 % through diet. The experiment was terminated 14 weeks after carcinogen administration. At autopsy, mammary tumours were removed and prepared for histopathological and immunohistochemical analysis. Moreover, in vitro evaluation in MCF-7 cells was carried out. Low-dose ORE suppressed tumour frequency by 55.5 %, tumour incidence by 44 %, and tumour volume by 44.5 % compared to control animals. Analysis of rat tumour cells showed Ki67, VEGFR-2, CD24, and EpCAM expression decrease and caspase-3 expression increase after low-dose ORE treatment. High-dose ORE lengthened tumour latency by 12.5 days; moreover, Bcl-2, VEGFR-2, CD24, and EpCAM expression decrease and caspase-3 expression increase in carcinoma cells were observed. Histopathological analysis revealed a decrease in the ratio of high-/low-grade carcinomas in both treated groups. In vitro studies showed that ORE decreased survival and proliferation of MCF-7 cells. In ORE-treated MCF-7 cells, an increase in cells expressing sub-G 0/G 1 DNA content and an increase in the percentage of annexin V/PI positive MCF-7 cells were observed. In vitro, both caspase-dependent and possible non-caspase-dependent apoptotic pathways were found. The deactivation of anti-apoptotic activity of Bcl-2, a decrease in mitochondrial membrane potential, and the activation of mitochondrial apoptosis pathway were observed in the ORE-treated MCF-7 cells. Our results demonstrate, for the first time, a distinct tumour-suppressive effect of oregano in the breast cancer model.

Aims Chalcones, naturally occurring open-chain polyphenols abundant in plants, have demonstrated antiproliferative activity in several cancer cell lines. In the present study, the potential anticancer activity of two synthetic analogues named Ch1 and Ch2 in colon cancer cell line was investigated. Main methods Antiproliferative activities of both synthetic analogues were assessed by Growth Inhibition Assay (MTT) and xCELLigence cell analysis. Apoptosis was assessed by annexin V/PI staining (early stage) or by DNA fragmentation (final stage). To study the cell death mechanism induced by tested substances, we assessed a series of assays including measurements of the caspase 3 activity, membrane mitochondrial potential (MMP) changes, reactive oxygen species (ROS) production by flow cytometry and expression of important apoptosis-related genes by realtime PCR. Key findings We found concentration and time-dependent cytotoxicity, inhibition of proliferation of Caco-2 cells after Ch1 and Ch2 treatment in parallel with G2/M phase cell cycle arrest and increased cell proportion in subG0/G1 population with annexin V positivity. We demonstrated that both Ch1 and Ch2 induced caspase-dependent cell death associated with increased ROS production, suppressed Bcl-2 and Bcl-xL and enhanced Bax expression. Treatment of Ch1 also suppressed α-, α1- and β5-tubulins, on the other hand Ch2 only suppressed α-tubulin expression. Significance Presented chalcones induce apoptosis by intrinsic pathways, and therefore may be an interesting strategy for cancer therapy.

Objectives: There has been considerable interest in both clinical and preclinical research about the role of phytochemicals in the reduction of risk for cancer in humans. The aim of this study was to determine the antineoplastic effects of Chlorella pyrenoidosa in experimental breast cancer in vivo and in vitro. Methods: In this experiment, the antineoplastic effects of Chlorella pyrenoidosa in the chemoprevention of N-methyl-N-nitrosourea-induced mammary carcinogenesis in female rats were evaluated. Chlorella powder was administered through diet at concentrations of 0.3% and 3%. The experiment was terminated 14 wk after carcinogen administration. At autopsy, mammary tumors were removed and prepared for histopathological and immunohistochemical analysis. In vitro cytotoxicity assay, parameters of apoptosis, and proliferation after chlorella treatment in human breast adenocarcinoma (MCF-7) cells were carried out. Results: Basic parameters of experimental carcinogenesis, mechanism of action (biomarkers of apoptosis, proliferation, and angiogenesis), chosen metabolic variables, and side effects after long-term chlorella treatment in animals were assessed. Chlorella at higher concentration suppressed tumor frequency by 61% (P < 0.02) and lengthened tumor latency by 12.5 d (P < 0.02) in comparison with the controls. Immunohistochemical analysis of rat tumor cells showed caspase-7 expression increase by 73.5% (P < 0.001) and vascular endothelial growth factor receptor-2 expression decrease by 19% (P = 0.07) after chlorella treatment. In a parallel in vitro study, chlorella significantly decreased survival of MCF-7 cells in a dose-dependent manner. In chlorella-treated MCF-7 cells, a significant increase in cells having sub-G(0)/G(1) DNA content and significant increase of early apoptotic and late apoptotic/necrotic cells after annexin V/PI staining assay were found. Decreases in mitochondrial membrane potential and increasing reactive oxygen species generation were observed in the chlorella-treated MCF-7 cells. Conclusions: This study is the first report on the antineoplastic effects of Chlorella pyrenoidosa in experimental breast cancer in vivo and in vitro. (C) 2015 Elsevier Inc. All rights reserved.

Lichens are a known source of approximately 800 unique secondary metabolites, many of which play important ecological roles, including regulating the equilibrium between symbionts. However, only a few of these compounds have been assessed for their effectiveness against various in vitro cancer models. Moreover, the mechanisms of biological activity of lichen secondary metabolites on living cells (including cancer cells) are still almost entirely unknown. In the present study, we investigated the mechanisms of cytotoxicity of four lichen secondary metabolites (parietin, atranorin, usnic acid and gyrophoric acid) on A2780 and HT-29 cancer cell lines. We found that usnic acid and atranorin were more effective anti-cancer compounds when compared to parietin and gyrophoric acid. Usnic acid and atranorin were capable of inducing a massive loss in the mitochondrial membrane potential, along with caspase-3 activation (only in HT-29 cells) and phosphatidylserine externalization in both tested cell lines. Induction of both ROS and especially RNS may be responsible, at least in part, for the cytotoxic effects of the tested compounds. Based on the detection of protein expression (PARP, p53, Bcl-2/Bcl-xL, Bax, p38, pp38) we found that usnic acid and atranorin are activators of programmed cell death in A2780 and HT-29, probably through the mitochondrial pathway.

Current treatment of breast cancer is often affected by resistance to therapeutics, for which the human epidermal growth factor receptor 2 (HER2) may be responsible. Here, we report for the first time the use of hypericin-mediated photodynamic therapy (HY-PDT) in combination with a selective HER2 inhibitor (AG 825) on SKBR-3, a HER2 overexpressing human breast adenocarcinoma cell line. The results demonstrate that HY-PDT is able to degrade HER2 with an impact on its signaling cascade. Combination with AG 825 resulted in increased apoptosis induction, total degradation of HER2 and inhibition of colony formation. Downregulation of HSP90, Mcl-1, Bcl-xL and upregulation of Bax was also observed. This knowledge provides the basis for the possible application of HY-PDT in preclinical and clinical models of breast cancer treatment.

Photodynamic therapy with hypericin (HY-PDT) is known as an efficient modality for treatment of various cancerous and non-cancerous diseases. Although the role of p53 protein in cell death signaling is well established, relatively little is known of its impact on the efficiency of HY-PDT. Comparison of sensitivity and long-term survival of p53-null versus wt-p53-expressing HCT-116 cells is reported here. The lack of p53 function did not affect cell proliferation or attenuate the initial phases of programmed cell death. However, analyses of apoptosis in the final stages revealed suppression of its incidence and delayed activation of caspase-3 in p53-null cells. Significantly higher clonogenic ability, especially in hypoxia, was identified in the case of p53-null cells. Induction of Mcl-1 and Bax levels were more prominent in wt-pt53 cells. Interestingly, the level of Bcl-2 did not react to HY-PDT at all, in both cell lines. Bringing the evidence together, we prove that despite insignificant impact on overall toxicity, expression of p53 affects the clonogenic efficiency of HCT-116 cells. Since destruction of tumor tissue and its vascular system by PDT tends to lead to hypoxia, superior survival of p53-deficient tumor cells under given conditions might result in recurrence of cancer diseases.