ABSTRACT
Chemotherapy drugs action against cancer is not selective, lead to adverse reactions and drug resistance. Combination therapies have proven more effective in defeating cancers. We hypothesize that plant extract/fraction contains many/several compounds and as such can target multiple pathways as cytotoxic agent and may also have chemo sensitizing activities. We designed a study in which, Asteriscus graveolens (Forssk.) Less (A. graveolens)-derived fraction that contains sesquiterpene lactone asteriscunolide isomers (AS) will be tested in combination with known chemotherapy drugs. Successful combination will permit to reduce chemotherapy drugs concentration and still get the same impact on cancer cells. Sesquiterpene lactone such as asteriscunolide isomers is a naturally occurring compound found in a variety of fruits, vegetables, and medicinal plants with anti-cancer properties. The experiments presented here showed that adding plant fraction containing AS permit reducing the concentration of cisplatin/etoposide/doxorubicin in order to reduce mouse BS-24-1 lymphoma cells (BS-24-1 cells) survival. It involved enhancing the production of Reactive Oxygen Species (ROS), activation of caspase-3 and inhibition of Topoisomerase I activity. Taken together, the results suggest that A. graveolens fraction sensitized BS-24-1 cells to cisplatin/etoposide/doxorubicin through induction of ROS and caspase-3-dependent apoptosis.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Antineoplastic Agents/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Asteraceae/chemistry , Cell Proliferation/drug effects , Lactones/pharmacology , Lymphoma/drug therapy , Sesquiterpenes/pharmacology , Animals , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/isolation & purification , Apoptosis/drug effects , Caspase 3/metabolism , Cell Cycle/drug effects , Cell Line, Tumor , Cisplatin/pharmacology , Doxorubicin/pharmacology , Drug Synergism , Etoposide/pharmacology , Isomerism , Lactones/chemistry , Lactones/isolation & purification , Lymphoma/metabolism , Lymphoma/pathology , Mice , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Plant Extracts/pharmacology , Sesquiterpenes/chemistry , Sesquiterpenes/isolation & purificationABSTRACT
Asteriscus graveolens (A. graveolens) plants contain among other metabolites, sesquiterpene lactone asteriscunolide isomers (AS). The crude extract and its fractions affected the viability of mouse BS-24-1 lymphoma cells (BS-24-1 cells) with an IC50 of 3 µg/mL. The fraction was cytotoxic to cancer cells but not to non-cancerous cells (human induced pluripotent stem cells); its activity was accompanied by a concentration- and time-dependent appearance of apoptosis as determined by DNA fragmentation and caspase-3 activity. High levels of Reactive Oxygen Species (ROS) were rapidly observed (less than 1 min) after addition of the fraction followed by an increase in caspase-3 activity three hours later. Comparison of RNA-seq transcriptome profiles from pre-and post-treatment of BS-24-1 cells with crude extract of A. graveolens yielded a list of 2293 genes whose expression was significantly affected. This gene set included genes encoding proteins involved in cell cycle arrest, protection against ROS, and activation of the tumor suppressor P53 pathway, supporting the biochemical findings on ROS species-dependent apoptosis induced by A. graveolens fraction. Interestingly, several of the pathways and genes affected by A. graveolens extract are expressed following treatment of human cancer cells with chemotherapy drugs. We suggest, that A. graveolens extracts maybe further developed into selective chemotherapy.
Subject(s)
Antineoplastic Agents/pharmacology , Asteraceae/chemistry , DNA Fragmentation/drug effects , Gene Expression Regulation/drug effects , Induced Pluripotent Stem Cells/drug effects , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/chemistry , Apoptosis/drug effects , Caspase 3/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Dose-Response Relationship, Drug , Humans , Mice , Plant Extracts/administration & dosage , Plant Extracts/chemistry , Plant Extracts/pharmacology , Plant Leaves/chemistry , Reactive Oxygen Species/metabolism , Sesquiterpenes/administration & dosage , Sesquiterpenes/chemistry , Sesquiterpenes/pharmacology , TranscriptomeABSTRACT
Protection of stalled replication forks is essential to prevent genome instability, a major driving force of tumorigenesis. Several key regulators of DNA double-stranded break (DSB) repair, including 53BP1 and RIF1, have been implicated in fork protection. MAD2L2, also known as REV7, plays an important role downstream of 53BP1/RIF1 by counteracting resection at DSBs in the recently discovered shieldin complex. The ability to bind and counteract resection at exposed DNA ends at DSBs makes MAD2L2/shieldin a prime candidate for also suppressing nucleolytic processing at stalled replication forks. However, the function of MAD2L2/shieldin outside of DNA repair is unknown. Here we address this by using genetic and single-molecule analyses and find that MAD2L2 is required for protecting and restarting stalled replication forks. MAD2L2 loss leads to uncontrolled MRE11-dependent resection of stalled forks and single-stranded DNA accumulation, which causes irreparable genomic damage. Unexpectedly, MAD2L2 limits resection at stalled forks independently of shieldin, since fork protection remained unaffected by shieldin loss. Instead, MAD2L2 cooperates with the DNA polymerases REV3L and REV1 to promote fork stability. Thus, MAD2L2 suppresses aberrant nucleolytic processing both at DSBs and stalled replication forks by differentially engaging shieldin and REV1/REV3L, respectively.