Kholoud Arafat

Inhibition of cell survival, invasion, tumor growth and histone deacetylase activity by the dietary flavonoid luteolin in human epithelioid cancer cells

Phytochemical compounds and histone deacetylase (HDAC) inhibitors are emerging as a new generation of anticancer agents with limited toxicity in cancer patients. We investigated the impact of luteolin, a dietary flavonoid, on survival, migration, invasion of cancer cells in vitro, and tumor growth in vivo. Luteolin (25-200μM) decreased the viability of human cancer cell lines originating from the lung (LNM35), colon (HT29), liver (HepG2) and breast (MCF7/6 and MDA-MB231-1833). Luteolin effectively increased the sub-G1 (apoptotic) fraction of cells through caspase-3 and -7 dependent pathways. We provide evidence that luteolin at sub-lethal/non-toxic concentrations inhibited the invasive potential of LNM35, MCF-7/6 and MDA-MB231-1833 cancer cells using Matrigel as well as the chick heart and Oris invasion assays. Moreover, we demonstrate for the first time that luteolin is a potent HDAC inhibitor that potentiates the cytotoxicity of cisplatin in LNM35 cells and decreases the growth of LNM35 tumor xenografts in athymic mice after intraperitoneal injection (20mg/kg/day for 18days) Thus, luteolin, in combination with standard anticancer drugs such as cisplatin, may be a promising HDAC inhibitor for the treatment of lung cancer.

Thymoquinone as an anticancer agent: evidence from inhibition of cancer cells viability and invasion in vitro and tumor growth in vivo

Phytochemical compounds are emerging as a new generation of anticancer agents with limited toxicity in cancer patients. The purpose of this study was to investigate the potential impact of thymoquinone (TQ), the major constituent of black seed, on survival, invasion of cancer cells in vitro, and tumor growth in vivo. Exposure of cells derived from lung (LNM35), liver (HepG2), colon (HT29), melanoma (MDA‐MB‐435), and breast (MDA‐MB‐231 and MCF‐7) tumors to increasing TQ concentrations resulted in a significant inhibition of viability through the inhibition of Akt phosphorylation leading to DNA damage and activation of the mitochondrial‐signaling proapoptotic pathway. We provide evidence that TQ at non‐toxic concentrations inhibited the invasive potential of LNM35, MDA‐MB‐231, and MDA‐MB231‐1833 cancer cells. Moreover, we demonstrate that TQ synergizes with DNA‐damaging agent cisplatin to inhibit cellular viability. The anticancer activity of thymoquinone was also investigated in athymic mice inoculated with the LNM35 lung cells. Administration of TQ (10 mg/kg/i.p.) for 18 days inhibited the LNM35 tumor growth by 39% (P < 0.05). Tumor growth inhibition was associated with significant increase in the activated caspase‐3. The in silico target identification suggests several potential targets of TQ mainly HDAC2 proteins and the 15‐hydroxyprostaglandin dehydrogenase. In this context, we demonstrated that TQ treatment resulted in a significant inhibition of HDAC2 proteins. In view of the available experimental findings, we contend that thymoquinone and/or its analogues may have clinical potential as an anticancer agent alone or in combination with chemotherapeutic drugs such as cisplatin.

Carnosol induces ROS-mediated beclin1-independent autophagy and apoptosis in triple negative breast cancer

Background In this study we investigated the in vitro and in vivo anticancer effect of carnosol, a naturally occurring polyphenol, in triple negative breast cancer. Results We found that carnosol significantly inhibited the viability and colony growth induced G2 arrest in the triple negative MDA-MB-231. Blockade of the cell cycle was associated with increased p21/WAF1 expression and downregulation of p27. Interestingly, carnosol was found to induce beclin1-independent autophagy and apoptosis in MDA-MB-231 cells. The coexistence of both events, autophagy and apoptosis, was confirmed by electron micrography. Induction of autophagy was found to be an early event, detected within 3 h post-treatment, which subsequently led to apoptosis. Carnosol treatment also caused a dose-dependent increase in the levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2). Moreover, we show that carnosol induced DNA damage, reduced the mitochondrial potential and triggered the activation of the intrinsic and extrinsic apoptotic pathway. Furthermore, we found that carnosol induced a dose-dependent generation of reactive oxygen species (ROS) and inhibition of ROS by tiron, a ROS scavenger, blocked the induction of autophagy and apoptosis and attenuated DNA damage. To our knowledge, this is the first report to identify the induction of autophagy by carnosol. Conclusion In conclusion our findings provide strong evidence that carnosol may be an alternative therapeutic candidate against the aggressive form of breast cancer and hence deserves more exploration.

Frondoside A inhibits human breast cancer cell survival, migration, invasion and the growth of breast tumor xenografts.

Breast cancer is a major challenge for pharmacologists to develop new drugs to improve the survival of cancer patients. Frondoside A is a triterpenoid glycoside isolated from the sea cucumber, Cucumaria frondosa. It has been demonstrated that Frondoside A inhibited the growth of pancreatic cancer cells in vitro and in vivo. We investigated the impact of Frondoside A on human breast cancer cell survival, migration and invasion in vitro, and on tumor growth in nude mice, using the human estrogen receptor-negative breast cancer cell line MDA-MB-231. The non-tumorigenic MCF10-A cell line derived from normal human mammary epithelium was used as control. Frondoside A (0.01-5 μM) decreased the viability of breast cancer cells in a concentration- and time-dependent manner, with 50%-effective concentration (EC50) of 2.5 μM at 24h. MCF10-A cells were more resistant to the cytotoxic effect of Frondoside A (EC50 superior to 5 μM at 24 h). In the MDA-MB-231 cells, Frondoside A effectively increased the sub-G1 (apoptotic) cell fraction through the activation of p53, and subsequently the caspases 9 and 3/7 cell death pathways. In addition, Frondoside A induced a concentration-dependent inhibition of MDA-MB-231 cell migration and invasion. In vivo, Frondoside A (100 μg/kg/dayi.p. for 24 days) strongly decreased the growth of MDA-MB-231 tumor xenografts in athymic mice, without manifest toxic side-effects. Moreover, we found that Frondoside A could enhance the killing of breast cancer cells induced by the chemotherapeutic agent paclitaxel. These findings identify Frondoside A as a promising novel therapeutic agent for breast cancer.

Frondoside A Suppressive Effects on Lung Cancer Survival, Tumor Growth, Angiogenesis, Invasion, and Metastasis

A major challenge for oncologists and pharmacologists is to develop less toxic drugs that will improve the survival of lung cancer patients. Frondoside A is a triterpenoid glycoside isolated from the sea cucumber, Cucumaria frondosa and was shown to be a highly safe compound. We investigated the impact of Frondoside A on survival, migration and invasion in vitro, and on tumor growth, metastasis and angiogenesis in vivo alone and in combination with cisplatin. Frondoside A caused concentration-dependent reduction in viability of LNM35, A549, NCI-H460-Luc2, MDA-MB-435, MCF-7, and HepG2 over 24 hours through a caspase 3/7-dependent cell death pathway. The IC50 concentrations (producing half-maximal inhibition) at 24 h were between 1.7 and 2.5 µM of Frondoside A. In addition, Frondoside A induced a time- and concentration-dependent inhibition of cell migration, invasion and angiogenesis in vitro. Frondoside A (0.01 and 1 mg/kg/day i.p. for 25 days) significantly decreased the growth, the angiogenesis and lymph node metastasis of LNM35 tumor xenografts in athymic mice, without obvious toxic side-effects. Frondoside A (0.1–0.5 µM) also significantly prevented basal and bFGF induced angiogenesis in the CAM angiogenesis assay. Moreover, Frondoside A enhanced the inhibition of lung tumor growth induced by the chemotherapeutic agent cisplatin. These findings identify Frondoside A as a promising novel therapeutic agent for lung cancer.

Salinomycin induces apoptosis and senescence in breast cancer: upregulation of p21, downregulation of survivin and histone H3 and H4 hyperacetylation.

BACKGROUND In the present study, we investigated the effect of Salinomycin on the survival of three human breast cancer cell lines MCF-7, T47D and MDA-MB-231 grown in adherent culture conditions. METHODS Cell viability was measured by Cell Titer-Glo and Trypan blue exclusion assay. Apoptosis was determined by caspase 3/7 activation, PARP cleavage and Annexin V staining. Cell cycle distribution was assessed by propidium iodide flow cytometry. Senescence was confirmed by measuring the senescence-associated β-galactosidase activity. Changes in protein expression and histone hyperacetylation was determined by western blot and confirmed by immunofluorescence assay. RESULTS Salinomycin was able to inhibit the growth of the three cell lines in time- and concentration-dependent manners. We showed that depending on the concentrations used, Salinomycin elicits different effects on the MDA-MB-231 cells. High concentrations of Salinomycin induced a G2 arrest, downregulation of survivin and triggered apoptosis. Interestingly, treatment with low concentrations of Salinomycin induced a transient G1 arrest at earlier time point and G2 arrest at later point and senescence associated with enlarged cellmorphology, upregulation of p21 protein, increase in histone H3 and H4 hyperacetylation and expression of SA-β-Gal activity. Furthermore, we found that Salinomycin was able to potentiate the killing of the MCF-7 and MDA-MB-231 cells, by the chemotherapeutic agents, 4-Hydroxytamoxifen and frondo side A, respectively. CONCLUSION Our data are the first to link senescence and histone modifications to Salinomycin. SIGNIFICANCE This study provides a new insight to better understand the mechanism of action of Salinomycin, at least in breast cancer cells.

In vitro oxime protection of human red blood cell acetylcholinesterase inhibited by diisopropyl-fluorophosphate.

Oximes are enzyme reactivators used in treating poisoning with organophosphorus cholinesterase (AChE) inhibitors. The oxime dose which can be safely administered is limited by the intrinsic toxicity of the substances such as their own AChE‐inhibiting tendency. Clinical experience with the available oximes is disappointing. To meet this need, new AChE reactivators of potential clinical utility have been developed. The purpose of the study was to estimate in vitro both the intrinsic toxicity and the extent of possible protection conferred by established (pralidoxime, obidoxime, HI‐6, methoxime, trimedoxime) and experimental (K‐type) oximes, using diisopropyl‐fluoro‐phosphate (DFP) as an AChE inhibitor. The IC50 of DFP against human red blood cell AChE was determined (∼120 nm). Measurements were then repeated in the presence of increasing oxime concentrations, leading to an apparent increase in DFP IC50. Calculated IC50 values were plotted against oxime concentrations to obtain an IC50 shift curve. The slope of this shift curve (tanα) was used to quantify the magnitude of the protective effect (nm IC50 increase per µm oxime). We show that, in the case of a linear relationship between oxime concentration and IC50, the binding constant K, determined using the Schild equation, equals IC50/DFP/tanα. Based on the values of tanα and of the binding constant K, some of the new K‐oxime reactivators are far superior to pralidoxime (tanα = 0.8), obidoxime (1.5), HI‐6 (0.8), trimedoxime (2.9) and methoxime (5.9), with K‐107 (17), K‐108 (20), and K‐113 (16) being the outstanding compounds. Copyright

Inhibitory Effects of Salinomycin on Cell Survival, Colony Growth, Migration, and Invasion of Human Non-Small Cell Lung Cancer A549 and LNM35: Involvement of NAG-1.

A major challenge for oncologists and pharmacologists is to develop more potent and less toxic drugs that will decrease the tumor growth and improve the survival of lung cancer patients. Salinomycin is a polyether antibiotic used to kill gram-positive bacteria including mycobacteria, protozoans such as plasmodium falciparum, and the parasites responsible for the poultry disease coccidiosis. This old agent is now a serious anti-cancer drug candidate that selectively inhibits the growth of cancer stem cells. We investigated the impact of salinomycin on survival, colony growth, migration and invasion of the differentiated human non-small cell lung cancer lines LNM35 and A549. Salinomycin caused concentration- and time-dependent reduction in viability of LNM35 and A549 cells through a caspase 3/7-associated cell death pathway. Similarly, salinomycin (2.5–5 µM for 7 days) significantly decreased the growth of LNM35 and A549 colonies in soft agar. Metastasis is the main cause of death related to lung cancer. In this context, salinomycin induced a time- and concentration-dependent inhibition of cell migration and invasion. We also demonstrated for the first time that salinomycin induced a marked increase in the expression of the pro-apoptotic protein NAG-1 leading to the inhibition of lung cancer cell invasion but not cell survival. These findings identify salinomycin as a promising novel therapeutic agent for lung cancer.

Anti-Metastatic and Anti-Tumor Growth Effects of Origanum majorana on Highly Metastatic Human Breast Cancer Cells: Inhibition of NFκB Signaling and Reduction of Nitric Oxide Production

Background We have recently reported that Origanum majorana exhibits anticancer activity by promoting cell cycle arrest and apoptosis of the metastatic MDA-MB-231 breast cancer cell line. Here, we extended our study by investigating the effect of O . majorana on the migration, invasion and tumor growth of these cells. Results We demonstrate that non-cytotoxic concentrations of O . majorana significantly inhibited the migration and invasion of the MDA-MB-231 cells as shown by wound-healing and matrigel invasion assays. We also show that O . majorana induce homotypic aggregation of MDA-MB-231 associated with an upregulation of E-cadherin protein and promoter activity. Furthermore, we show that O . majorana decrease the adhesion of MDA-MB-231 to HUVECs and inhibits transendothelial migration of MDA-MB-231 through TNF-α-activated HUVECs. Gelatin zymography assay shows that O . majorana suppresses the activities of matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9). ELISA, RT-PCR and Western blot results revealed that O . majorana decreases the expression of MMP-2, MMP-9, urokinase plasminogen activator receptor (uPAR), ICAM-1 and VEGF. Further investigation revealed that O . majorana suppresses the phosphorylation of IκB, downregulates the nuclear level of NFκB and reduces Nitric Oxide (NO) production in MDA-MB-231 cells. Most importantly, by using chick embryo tumor growth assay, we also show that O . majorana promotes inhibition of tumor growth and metastasis in vivo. Conclusion Our findings identify Origanum majorana as a promising chemopreventive and therapeutic candidate that modulate breast cancer growth and metastasis.

Analogues of the frog skin peptide alyteserin-2a with enhanced antimicrobial activities against Gram-negative bacteria

The emergence of strains of multidrug‐resistant Gram‐negative bacteria mandates a search for new types of antimicrobial agents. Alyteserin‐2a (ILGKLLSTAAGLLSNL.NH2) is a cationic, α‐helical peptide, first isolated from skin secretions of the midwife toad, Alytes obstetricans, which displays relatively weak antimicrobial and haemolytic activities. Increasing the cationicity of alyteserin‐2a while maintaining amphipathicity by the substitution Gly11→ Lys enhanced the potency against both Gram‐negative and Gram‐positive bacteria by between fourfold and 16‐fold but concomitantly increased cytotoxic activity against human erythrocytes by sixfold (mean concentration of peptide producing 50% cell death; LC50 = 24 µm). Antimicrobial potency was increased further by the additional substitution Ser7→Lys, but the resulting analogue remained cytotoxic to erythrocytes (LC50 = 38 µm). However, the peptide containing d‐lysine at positions 7 and 11 showed high potency against a range of Gram‐negative bacteria, including multidrug‐resistant strains of Acinetobacter baumannii and Stenotrophomonas maltophilia (minimum inhibitory concentration = 8 µm) but appreciably lower haemolytic activity (LC50 = 185 µm) and cytotoxicity against A549 human alveolar basal epithelial cells (LC50 = 65 µm). The analogue shows potential for treatment of nosocomial pulmonary infections caused by bacteria that have developed resistance to commonly used antibiotics. Copyright