Anticancer effect of Dihydroartemisinin via dual control of ROS-induced apoptosis and protective autophagy in prostate cancer 22Rv1 cells.

BACKGROUND: Dihydroartemisinin (DHA), a natural agent, exhibits potent anticancer activity. However, its biological activity on prostate cancer (PCa) 22Rv1 cells has not been previously investigated. OBJECTIVE: In this study, we demonstrate that DHA induces anticancer effects through the induction of apoptosis and autophagy. METHODS: Cell viability and proliferation rate were assessed using the CCK-8 assay and cell clone formation assay. The generation of reactive oxygen species (ROS) was detected by flow cytometry. The molecular mechanism of DHA-induced apoptosis and autophagy was examined using Western blot and RT-qPCR. The formation of autophagosomes and the changes in autophagy flux were observed using transmission electron microscopy (TEM) and confocal microscopy. The effect of DHA combined with Chloroquine (CQ) was assessed using the EdU assay and flow cytometry. The expressions of ROS/AMPK/mTOR-related proteins were detected using Western blot. The interaction between Beclin-1 and Bcl-2 was examined using Co-IP. RESULTS: DHA inhibited 22Rv1 cell proliferation and induced apoptosis. DHA exerted its anti-prostate cancer effects by increasing ROS levels. DHA promoted autophagy progression in 22Rv1 cells. Inhibition of autophagy enhanced the pro-apoptotic effect of DHA. DHA-induced autophagy initiation depended on the ROS/AMPK/mTOR pathway. After DHA treatment, the impact of Beclin-1 on Bcl-2 was weakened, and its binding with Vps34 was enhanced. CONCLUSION: DHA induces apoptosis and autophagy in 22Rv1 cells. The underlying mechanism may involve the regulation of ROS/AMPK/mTOR signaling pathways and the interaction between Beclin-1 and Bcl-2 proteins. Additionally, the combination of DHA and CQ may enhance the efficacy of DHA in inhibiting tumor cell activity.

Dihydroartemisinin induces cell apoptosis through repression of UHRF1 in prostate cancer cells.

Prostate cancer (PCa) seriously jeopardizes men's health worldwide. Dihydroartemisinin, which is an effective antimalarial agent, has shown potential anticancer effects in various human cancer cell lines, including PCa cells. However, the mechanisms underlying the anticancer activity of dihydroartemisinin are not fully understood. Ubiquitin-like with plant homeodomain and ring finger domain 1 (UHRF1) is highly expressed in a variety of tumors and is negatively correlated with the prognosis of various tumors. We reported previously that UHRF1 is downregulated during apoptosis induced by dihydroartemisinin in PC-3 PCa cells. In this study, we transfected PC-3 cells with lentiviruses containing UHRF1 or shRNA-UHRF1. Then, the cells were treated with dihydroartemisinin at different concentrations. Our data showed that overexpression of UHRF1 promoted cell proliferation and migration in PC-3 cells, inhibited cell apoptosis, increased cell proportion in G2 phase, increased DNA methyltransferase 1 and decreased p16INK4A expression at mRNA and protein levels. Downregulation of UHRF1 produces the opposite results. Moreover, the phenomena caused by overexpression of UHRF1 were inhibited after dihydroartemisinin treatment. Compared with control cells, cells overexpressing UHRF1 can resist the proapoptotic and antiproliferative effects of dihydroartemisinin to a certain extent. The effects of UHRF1 knockdown were further aggravated by dihydroartemisinin treatment, but no statistically significant effect was observed with increasing drug concentration. Our results suggested that dihydroartemisinin decreases proliferation and migration but enhances apoptosis of PCa cells, likely by downregulating UHRF1 and upregulating p16INK4A.

Identification of miRNA signature associated with BMP2 and chemosensitivity of TMZ in glioblastoma stem-like cells.

Glioblastoma multiform (GBM) is the most lethal intracranial tumor in adults. Glioblastoma stem-like cells (GSCs) are responsible for tumorigenesis and chemotherapy resistance. BMPs are known to increase temozolomide (TMZ) response in GSCs, however, the intracellular molecular mechanism remains largely unknown. In this study, we built a GSC cell model called U87S, and performed RNA sequencing to identify differentially expressed (DE) miRNA profiles in U87S cells treated with BMP2, TMZ or combined BMP2 and TMZ respectively. Bioinformatics analysis revealed that most DE miRNAs were involved in the cancer pathways, suggesting their crucial roles in gliomagenesis. Eight miRNAs from RNA-seq were validated. Four out of these miRNAs (has-miR-199a-3p, hsa-miR-374b-5p, hsa-miR-320d, and hsa-miR-339-5p) were found significantly up-regulated in GBM tumor tissues. One of them, hsa-miR-199a-3p, was significantly correlated with the survival of GBM patients, and differentially expressed in U87S cells. Expression of hsa-miR-199a-3p was up-regulated by BMP. Overexpression of hsa-miR-199a-3p in U87S cells inhibited cell viability and enhanced the cytotoxicity of TMZ. And activation of BMP boosted the effect of hsa-miR-199a-3p on cell viability and TMZ-mediated cytotoxicity. Besides, expressions of five predicted targets of hsa-miR-199a-3p were evaluated. Four of them were differentially expressed in GBM tumors. And one of them, SLC22A18, was associated with the survival of GBM patients. In the end, a hsa-miR-199a-3p-mediated ceRNA network was constructed for the convenience of future study. Together, our data provided DE miRNA expression profiles associated with BMP2 and TMZ in GSCs, which might lead to finding out miRNA-based target therapies that specially target GSCs.

Effect of dihydroartemisinin on UHRF1 gene expression in human prostate cancer PC-3 cells.

As the second most common cancer in men around the world, prostate cancer is increasingly gaining more attention. Dihydroartemisinin (DHA) has been proven to be a promising anticancer agent in vitro as well as in vivo in accumulating data. However, the detailed mechanisms of how DHA action in human prostate cancer PC-3 cells remain elusive. This study aimed to investigate the effects of DHA, a novel anticancer agent, by inhibiting the expression of ubiquitin like containing PHD and ring finger 1 (UHRF1) in PC-3 cells. The apoptosis and cell-cycle distribution were detected by flow cytometry. Quantitative real-time PCR was performed to examine both UHRF1 and DNA methyltransferase 1 (DNMT1) expressions at mRNA levels, whereas the expressions of UHRF1, DNMT1, and p16 proteins at protein levels were detected by Western blotting. Methylation levels of p16 CpG islands were determined by bisulfite genomic sequencing. We showed that DHA induced the downregulation of UHRF1 and DNMT1, accompanied by an upregulation of p16 in PC-3 cells. Decreased p16 promoter methylation levels in DHA-treated groups were also observed in PC-3 cells. Furthermore, DHA significantly induced apoptosis and G1/S cell-cycle arrest in PC-3 cells. Our results suggested that downregulation of UHRF1/DNMT1 is upstream to many cellular events, including G1 cell arrest, demethylation of p16, and apoptosis. Together, our study provides new evidence that DHA may serve as a potential therapeutic agent in the treatment of prostate cancer.

Mechanism of dihydroartemisinin-induced apoptosis in prostate cancer PC3 cells: An iTRAQ-based proteomic analysis.

AIMS: Prostate cancer (PCa) is one of the most common cancers in men in the world. Advanced PCa, especially castration-resistant PCa (CRPC), is difficult to cure. There is an urgent need to develop novel agents for CPRC. Dihydroartemisinin (DHA) is a semisynthetic derivative of artemisinin and is a well-known antimalarial drug. DHA has been documented to be a potential anticancer agent for PCa. However, the mechanisms underlying the anticancer activity of DHA are still unknown. MAIN METHODS: Proteomics analysis based on iTRAQ technology was performed to determine the protein profile changes in human prostate cancer PC3 cells treated by DHA, and apoptosis was detected by flow cytometry and transmission electron microscopy. KEY FINDINGS: DHA induced obvious apoptosis in PC3 cells. Using iTRAQ technology, we found 86 differentially expressed proteins linked to the cytotoxicity of DHA in PC3 cells. Gene ontology analysis showed the differentially expressed proteins were mainly associated with the protein synthesis and translation. Protein interaction network analysis and KEGG pathway analysis revealed altered aminoacyl-tRNA biosynthesis and metabolic pathways. Moreover, one candidate protein, heat shock protein HSP70 (HSPA1A), was identified by western blot analysis. SIGNIFICANCE: Our results indicate that multiple mechanisms involved in the anticancer activity of DHA in PC3 cells. Decreased HSP70 expression may have an important role in DHA-induced apoptosis in PC3 cells. Our data also provide novel insights into the anticancer mechanisms of DHA.

Dihydroartemisinin induces endoplasmic reticulum stress-mediated apoptosis in HepG2 human hepatoma cells.

AIMS AND BACKGROUND: Previous studies showed that dihydroartemisinin (DHA) possessed antitumor activity in many human tumor cells through the induction of apoptosis. The aim of this study was to investigate the effects of DHA on apoptosis in the human hepatocellular carcinoma cell line HepG2 and the possible molecular mechanisms involved. METHODS: The inhibitory effect of DHA on HepG2 cells was measured by MTT assay. The percentage of apoptotic cells was detected by flow cytometry with double staining of fluorescein isothiocyanate-annexin V/propidium iodide. The intracellular production of reactive oxygen species (ROS) and intracellular Ca2+ concentration ([Ca2+]i) were detected by fluorescence spectrophotometry. Protein expression of GADD153, Bcl-2 and Bax in HepG2 cells was examined by Western blot and immunocytochemistry. RESULTS: DHA significantly inhibited proliferation of HepG2 cells in a dose- and time-dependent manner. The apoptosis rates in HepG2 cells treated with 0, 50, 100 and 200 µmol/L DHA for 24 hours were 2.53 ± 0.88%, 24.85 ± 3.63%, 35.27 ± 5.92% and 48.53 ± 7.76%, respectively. Compared with the control group, DHA significantly increased ROS generation and [Ca2+]i level (P <0.05), with the generation of ROS preceding the increase in [Ca2+]i. An increase in GADD153 and Bax expression and a decrease in Bcl-2 were observed in DHA-treated cells. Pretreatment with the antioxidant N-acetylcysteine could attenuate the effects of DHA in the experiments. CONCLUSION: DHA could inhibit proliferation and induce apoptosis in HepG2 cell lines through increasing the intracellular production of ROS and [Ca2+]i. Endoplasmic reticulum stress-induced apoptosis may contribute to this effect by regulating the expression of GADD153, proapoptotic Bax, and antiapoptotic Bcl-2.

[Clinical significance of histopathologic and ultrastructural pathologic examination in etiological diagnosis of infantile cholestatic diseases].

OBJECTIVE: To study the features of histopathologic and ultrastructural pathologic changes of liver biopsy in patients with infantile cholestatic disease, and to investigate its diagnostic significance combining with the clinical data. METHODS: Thirty-six children diagnosed as infantile cholestatic disease and received liver biopsy in Chongqing Medical University Children's Hospital from Jun 2007 to Oct 2008 were enrolled and the pathologic and ultrastructural pathologic changes of liver were analyzed. RESULTS: Morphologic changes under light microscope in liver tissues included hepatocyte swelling, hepatocyte denaturation, hepatocyte necrosis, multinucleated giant cell formation, bile duct proliferation, fiber tissues proliferation and inflammatory cells infiltration in liver lobules and portal regions. The characteristics of cholestasis including intralobular cholestasis, acinus formation, feather-like cytoplasmic filaments and bile stasis in bile canaliculi were observed. The morphologic changes of biliary atresia were observed in 7 cases whose image investigations showed no obstruction of biliary tract. Nuclear changes, resolution of cytoplasm, inflammatory cell infiltration, collagen fiber proliferation and increased number of lysosomes were observed under electromicroscope. Two cases of glycogen storage disease, 1 case of Niemann-Pick disease and 1 case of lipid storage disease with unknown cause were confirmed by the combination of histological changes and clinical manifestations. CONCLUSION: Common pathologic changes of liver tissues existed under light microscope or electroscope. The diagnosis of hereditary metabolic disorders could be made increasingly by application of these two technologies in clinical practice. It is difficult to diagnose biliary atresia in early childhood by image investigations and the pathological changes of liver tissues are helpful.