Anti-cancer activity of phytochemicals extracted from Urtica pilulifera on Hela cells.

Urtica pilulifera is effective against cancer cell growth but its bioactive compounds and the mechanism of action behind its effect are still to be clarified. This study evaluated the anticancer activity of compounds extracted from Urtica pilulifera leaves against cervical cancer cells. The cytotoxic effect of water, methanol and hexane extracts of Urtica pilulifera leaves was assessed by MTT assay. The most potent extract was fractionated by column chromatography and its anticancer activity was evaluated. GC-MS was used to identify the chemical components of the different fractions. Western blotting analysis was used to determine the level of specific apoptotic and cell cycle protein markers. Data showed that the hexanic extract exhibits the most potent cytotoxicity against Hela cells. Fraction 4 of the hexanic extract showed the most potent antiproliferative effect against Hela cells. GC-MS analysis showed that the most prominent compound in fraction 4 is phytol. Fraction 4 treatment induced cell cycle arrest and intrinsic apoptosis in Hela cells as evident by the increasing levels of p21, p53, PARP cleavage and active caspase 9. These findings reveal the ability of U. pilulifera hexanic extract to induce cell cycle arrest and apoptosis in cervical cancer cells.

Design, Synthesis and Biological Evaluation of Novel Pyrazolo[1,2,4]triazolopyrimidine Derivatives as Potential Anticancer Agents.

Three novel pyrazolo-[4,3-e][1,2,4]triazolopyrimidine derivatives (1, 2, and 3) were designed, synthesized, and evaluated for their in vitro biological activity. All three compounds exhibited different levels of cytotoxicity against cervical and breast cancer cell lines. However, compound 1 showed the best antiproliferative activity against all tested tumor cell lines, including HCC1937 and HeLa cells, which express high levels of wild-type epidermal growth factor receptor (EGFR). Western blot analyses demonstrated that compound 1 inhibited the activation of EGFR, protein kinase B (Akt), and extracellular signal-regulated kinase (Erk)1/2 in breast and cervical cancer cells at concentrations of 7 and 11 µM, respectively. The results from docking experiments with EGFR suggested the binding of compound 1 at the ATP binding site of EGFR. Furthermore, the crystal structure of compound 3 (7-(4-bromophenyl)-9-(pyridin-4-yl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine) was determined by single crystal X-ray analysis. Our work represents a promising starting point for the development of a new series of compounds targeting EGFR.

Identifying Priorities and Needs to Improve Oncology Research in the Gaza Strip, Palestine.

Purpose: The purpose of this study was to (1) identify the priorities of oncology research in the Gaza Strip; (2) explore the needs for improving oncology research in the Gaza Strip, Palestine. Participants and Methods: A qualitative approach for data collection was used in this study. After obtaining the ethical approvals to conduct this study, a sample of 42 health-care providers who are involved in providing oncology care and research in the Gaza Strip were included in this study. Data were collected by the researchers through seven focus groups. Thematic coding was used for data analysis. Two main themes and several sub-themes were extracted during the data analysis. Results: The two main themes extracted from data analysis were research priorities and research needs. Participants identified several priorities in relation to oncology research that are assessing for cancer awareness, cancer prevention, exploring and finding new molecular biomarkers, screening for germ-line mutations related to the most common cancers, determining genetic and environmental risk factors for developing cancer, and exploring and testing new cancer therapies. Concerning research needs, participants identified several needs to enhance oncology research, which are financial needs, need for training, availability of data, creation of interdisciplinary research teams, and transforming in vitro studies to in vivo. Conclusion: Well-designed studies will certainly help to identify the priorities and needs to improve oncology research in the Gaza Strip, which is considered one of the most important steps to help push these priorities onto the agenda of health policymakers. Therefore, they will work to set goals and design policies and programs aiming to reduce incidence and prevalence rates of cancer in the Gaza Strip, promote early detection of cancer, improve prognosis, and reduce mortality related to cancer.

Synthesis and bioassay of 3-Aryl -1-(pyridin-4-yl)benzo[4,5]imidazo[1,2-d][1,2,4]- triazin-4(3H)-ones as anti-cancer agents.

Four novel 3-Aryl -1-(pyridin-4-yl)benzo[4,5]imidazo[1,2-d][1,2,4]- triazin-4(3H)-ones derivatives (C1 to C4) have been designed, synthesized, and evaluated for their anticancer activity. The structure of compounds was characterized by IR,1H NMR, 13C NMR and high-resolution mass (HRMS). The crystal structures of C1, C2 and C4 were previously determined by single-crystal X-ray analysis.The results from docking experiments with EGFR suggested the binding of the compounds at the active site of EGFR. The new compounds exhibited different levels of cytotoxicity against HCC1937 and MCF7 breast cancer cells. Results of the MTT assay identified C3 as the most cytotoxic of the series against both MCF7 and HCC1937 breast cancer cell lines with IC50 values of 36.4 and 48.2 µM, respectively. In addition to its ability to inhibit cell growth and colony formation ability, C3 also inhibited breast cancer cell migration. Western blotting results showed that C3 treatment inhibited EGFR signaling and induced cell cycle arrest and apoptosis as indicated by the low level of p-EGFR and p-AKT and the increasing levels of p53, p21 and cleaved PARP. Our work represents a promising starting point for the development of a new series of compounds targeting cancer cells.

The Anticancer Effects of Novel Imidazo[1,2-a]Pyridine Compounds against HCC1937 Breast Cancer Cells.

BACKGROUND: Anticancer drugs confront clinical obstacles such as drug resistance and adverse effects. Imidazo[1,2-a]pyridines (IPs) compounds have lately gained considerable interest as possible anticancer therapeutics due to their potent inhibitory function against cancers cells. This study was to determine the anticancer activities of three novel IPs (IP-5, IP-6, and IP-7) against the HCC1937 breast cancer cell line in vitro. MATERIALS AND METHODS: The cytotoxic and anti-proliferative effects of IPs compounds in HCC1937 cells were determined by cell viability (MTT) assay, trypan blue assay, and clonogenic survival assay. Scratch motility assay was used to test the antimigration ability of the IPs. Western blot analysis was carried out to detect the level of apoptosis and cell cycle protein markers and to understand the mechanism of action of IPs compounds. RESULTS: IP-5 and IP-6 have a strong cytotoxic impact against HCC1937 cells with IC50 values of 45µM and 47.7µM respectively. IP-7 possesses less cytotoxic effect against HCC1937 cells with IC50 of 79.6µM. Trypan blue assay showed that the three compounds induce significant cell death in the HCC1937 cells. Clonogenic and mammosphere assays demonstrated that IP-5 reduced the HCC1937 cells survival rate by more than 25.0% at 1000 cell concentrations. Western blotting analysis showed that IP-5 compound causes cell cycle arrest as noted by the increasing levels of p53 and p21 in treated cells. IP-5 induced an extrinsic apoptosis pathway as reveals from the increased activity of caspase 7, caspase 8, and the increasing level of PARP cleavage in treated cells. Also, IP-5 treated cells revealed segmented chromatin which is characteristic of apoptotic cells as shown by DAPI stain. Importantly, In comparison to control cells, IP-5-treated cells exhibited lower levels of pAKT. CONCLUSIONS: The novel three IPs compounds represent potential active anticancer compounds against HCC1937 breast cancer cells in vitro.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Overexpression of TBX3 transcription factor as a potential diagnostic marker for breast cancer.

The T-box 3 (TBX3) transcription factor has been shown to serve multiple roles in normal development. Recent findings have revealed that TBX3 is overexpressed in different types of carcinomas, including breast, cervical, ovarian, melanoma, pancreatic, lung, liver, bladder, head and neck. Therefore, the present study investigated the significance of TBX3 as a diagnostic marker of breast cancer. To achieve this aim, breast cancer samples and their adjacent normal tissues were collected from 51 breast cancer patients from the European Gaza hospital during 2015-2016. Sections from each sample were immune-stained by anti-TBX3 and suitable secondary and tertiary antibodies. TBX3 levels were evaluated in cancerous and normal samples. Clinicopathological data for each patient were documented. The correlation between TBX3 levels and the clinicopathological parameters were statistically tested. The results revealed that TBX3 is significantly overexpressed in breast cancer tissues when compared with normal tissues. Furthermore, TBX3 was mainly a cytoplasmic protein in normal and breast cancer tissues. Notably, TBX3 levels exhibited a sensitivity of 78.4%, specificity of 79.6%, accuracy of 79% and area under the curve of 0.791 (0.700-0.882) at a cut-off value=9 as breast cancer marker. However, no significant associations were observed between TBX3 levels and other breast cancer markers including oestrogen receptor, progesterone receptor, human epidermal growth factor receptor 2, cancer antigen 15-3 and breast cancer stages. Altogether, these results suggested that TBX3 overexpression may be a potential biomarker for breast cancer.

Novel imidazo[1,2-a]pyridine inhibits AKT/mTOR pathway and induces cell cycle arrest and apoptosis in melanoma and cervical cancer cells.

The present study aimed to investigate the anti-cancer activity of imidazo[1,2-a]pyridine 5-7 in the A375 and WM115 melanoma and HeLa cervical cancer cell lines. The viability of cancer cells was analyzed by the MTT assay. Apoptosis was quantified by flow cytometry following staining of the cells with AnnexinV/propidium iodide (PI). The cell cycle was evaluated by flow cytometry after staining of cells with PI. The three compounds inhibited the proliferation of all cells for half maximal inhibitory concentration ranging from 9.7 to 44.6 µM following 48-h treatment. In addition, all cancer cells were more sensitive to compound 6 compared with the other compounds. Treatment with compound 6 induced G2/M cell cycle arrest and a significant increased level of intrinsic apoptosis in all tested cells. Furthermore, compound 6 reduced the levels of phospho (p)-protein kinase B and p-mechanistic target of rapamycin, and increased levels of the cell cycle inhibitors p53 and p21 and of the apoptosis-associated proteins BCL2 associated X protein and active caspase-9. Silencing p53 in A375 melanoma cells reduced compound 6-induced apoptosis, which suggested that compound 6 may induce p53-partially mediated apoptosis. These results demonstrated that imidazo[1,2-a]pyridines 5-7 are potential effective compounds in the treatment of melanoma and cervical cancers.

Combined pitavastatin and dacarbazine treatment activates apoptosis and autophagy resulting in synergistic cytotoxicity in melanoma cells.

Melanoma is an aggressive skin cancer and its incidence is increasing faster than any other type of cancer. Whilst dacarbazine (DTIC) is the standard chemotherapy for metastatic melanoma, it has limited success. Statins, including pitavastatin, have been demonstrated to have a range of anti-cancer effects in a number of human cancer cell lines. The present study therefore explored the anti-cancer activity of combined DTIC and pitavastatin in A375 and WM115 human melanoma cells. Cell survival assays demonstrated that combined DTIC and pitavastatin treatment resulted in synergistic cell death. Cell cycle analyses further revealed that this combined treatment resulted in a G1 cell cycle arrest, as well as a sub-G1 population, indicative of apoptosis. Activation of apoptosis was confirmed by Annexin V-fluorescein isothiocyanate/propidium iodide double-staining and an increase in the levels of active caspase 3 and cleaved poly (ADP-ribose) polymerase. Furthermore, it was demonstrated that apoptosis occurs through the intrinsic pathway, evident from the release of cytochrome c. Finally, combined DTIC and pitavastatin treatment was demonstrated to also activate autophagy as part of a cell death mechanism. The present study provides novel evidence to suggest that the combined treatment of DTIC and pitavastatin may be effective in the treatment of melanoma.

The palladacycle, AJ-5, exhibits anti-tumour and anti-cancer stem cell activity in breast cancer cells.

Breast cancer is the most common malignancy amongst women worldwide but despite enormous efforts to address this problem, there is still limited success with most of the current therapeutic strategies. The current study describes the anti-cancer activity of a binuclear palladacycle complex (AJ-5) in oestrogen receptor positive (MCF7) and oestrogen receptor negative (MDA-MB-231) breast cancer cells as well as human breast cancer stem cells. AJ-5 is shown to induce DNA double strand breaks leading to intrinsic and extrinsic apoptosis and autophagy cell death pathways which are mediated by the p38 MAP kinase. This study provides evidence that AJ-5 is potentially an effective compound in the treatment of breast cancer.

A novel binuclear palladacycle complex inhibits melanoma growth in vitro and in vivo through apoptosis and autophagy.

Malignant melanoma is an aggressive skin cancer and it is reported to be the most treatment-resistant human cancer. Here we describe the anti-tumour activity of a novel binuclear palladacycle complex (AJ-5) in vertical growth phase (ME1402) and metastatic (WM1158) melanoma cell lines. We show that compared to normal control cell lines, AJ-5 is more effective in inhibiting the proliferation of ME1402 and WM1158 melanoma cells with IC50 values of 0.19 and 0.20µM, respectively. Flow cytometry analyses showed that AJ-5 induced apoptosis (sub-G1 peak) which was confirmed by Annexin V-FITC/propidium iodide double-staining, nuclear fragmentation and an increase in the levels of PARP cleavage. Furthermore, AJ-5 was shown to induce both intrinsic and extrinsic apoptotic pathways as measured by PUMA, Bax and active caspases. Interestingly, AJ-5 treatment also simultaneously induced the formation of autophagosomes and led to an increase in the autophagy markers LC3II and Beclin1. Inhibition of autophagy reduced AJ-5 cytotoxicity suggesting that AJ-5 induced autophagy was a cell death and not cell survival mechanism. Moreover we show that AJ-5 induces the ATM-CHK2 DNA damage pathway and that its anti-tumour function is mediated by the p38 and ERK1/2 signalling pathways. Importantly, AJ-5 treatment efficiently reduced tumour growth in melanoma bearing mice and induced high levels of autophagy and apoptosis markers. Together these findings suggest that AJ-5 may be an effective chemotherapeutic drug in the treatment of melanoma, a highly aggressive and intractable cancer.