Study On Anticancer Effects And Underlying Mechanisms Of An Alkaloid Chaetominine Produced By The Marine Microorganism

Chaetominine was a quinazolinone alkaloid isolated from secondary metabolites of Aspergillus fumigatus CY018. It has been reported that chaetominine was cytotoxic towards colon cancer cell line and leukemia cell line in vitro. However, the anticancer effect and related mechanisms were still unclear. This study investigated the anticancer effect of chaetominine on human cancer cell lines and the possible mechanism which underpin the regulation by chaetominine in detail. This study would be of great significance for the development and utilization of marine microbial resources.Aimed at uncontrolling cell growth, the ability to kill cancer cells of chaetominine was detected by MTT assays. The results show that the most significant inhibitory effect of chaetominine on cell growth occurred in human leukemia cell line K562, when the IC₅₀ values of chaetominine and the positive control 5-FU were 33.7±0.2 nM and 55.0±1.07 nM, respectively. But chaetominine showed little cytotoxicity towards normal blood cells at concentrations below 100 nM, which suggested that cancer cells were more sensitive to chaetominine.Since induction of apoptosis is a popular strategy for cancer therapy, Hoechst 33258 staining, Annexin V-FITC/PI dual staining, DNA ladder analysis were performed to determine whether chaetominine induced apoptotic cell death in K562 cells. The high apoptosis rates, morphological apoptotic features, and DNA fragmentation caused by chaetominine indicated that the cytotoxicity was partially caused by its pro-apoptotic effect. Following JC-1 and western blot analysis found that the Bax/Bcl-2 ratio was upregulated （from 0.3 to 8）, which was followed by a decrease in mitochondrial membrane potential, release of cytochrome c from mitochondria into the cytosol, and stimulation of Apaf-1. Furthermore, activation of caspase-9 and caspase-3, which are the main executers of the apoptotic process, was observed. These results demonstrated that chaetominine induced cell apoptosis via the mitochondrial pathway.This study also demonstrated that the inhibitory effect of CHA on cell growth was associated with cell cycle regulation mechanism. Under chaetominine treatment, K562 cells underwent S-phase arrest following flow cytometry analysis. Western blot and qRT-PCR analyses suggested that chaetominine treatment increased the expression of p-ATR and Chk1 while decreased Cdc25A expression. Additionally, both protein and gene expression levels of cyclin A and CDK2 were repressed. These results revealed the role of chaetominine in in the regulation of ATR/Cdc25A/Chkl expression. These proteins are important determinants in the initiation of S-phase arrest.In addition, this study investigated the effect and possible mechanisms of chaetominine on drug resistance. Cytotoxicity assays indicated that K562/Adr resistance to adriamycin （ADR） did not occur in the presence of chaetominine and that chaetominine increased chemosensitivity of K562/Adr to ADR. Data show that chaetominine enhanced ADR-induced apoptosis and intracellular ADR accumulation in K562/Adr cells. Accordingly, chaetominine induced apoptosis by upregulating ROS, pro-apoptotic Bax and downregulating anti-apoptotic Bcl-2. RTPCR and western-blot confirmed that chaetominine suppressed highly expressed MRP1 at mRNA and protein levels. But little obvious alternation of another drug transporter MDR1 mRNA was observed. Furthermore, inhibition of MRP1 by chaetominine relied on inhibiting Akt phosphorylation and nuclear Nrf2. In summary, chaetominine strongly reverses drug resistance by interfering with the PI3K/Akt/Nrf2 signaling, resulting in reduction of MRP1-mediated drug efflux and induction of Bax/Bcl-2-dependent apoptosis in an ADR-resistant K562/Adr leukemia cell line.This study reveals the anticancer effects of chaetominine in vitro are related to the induction of cell apoptosis and cell arrest at S phase. In addition, CHA could reverse drug resistance through inducing apoptosis and inhibiting MRP1 exxpression. These results suggest the potential of chaetominine as a candidate for leukemia therapy.