Aristolochic acid I promotes the invasion and migration of hepatocellular carcinoma cells by activating the C3a/C3aR complement system.

Aristolochic acid is an established human carcinogen. Previous reports have demonstrated a link between aristolochic acid exposure and liver cancer prevalence in Asia. The C3a/C3AR axis plays an essential role in regulating cancer cell migration and invasion. Here, we focused on the relationship between AA I-induced migration, invasion and epithelial-mesenchymal transition in HCC cells, as well as the possible role of the C3a/C3AR axis in these effects. HCC cells were exposed to different concentrations of AA I for 24 h. Cell migration and invasion abilities were evaluated with wound healing assays and Transwell invasion assays. The protein and mRNA expression levels were detected by western blot, immunofluorescence and quantitative real-time polymerase chain reaction (qRT-PCR) assays. Furthermore, the level of complement component C3a in the cell supernatant was determined by enzyme-linked immunosorbent assay. C3aRA, a C3a receptor antagonist, was used to block the C3a-C3aR axis. The results showed that aristolochic acid I promoted HCC cell invasion and migration. AAI exposure also induced EMT in HCC cells through E-cadherin downregulation and Snail, N-cadherin, and vimentin upregulation. AAI exposure increased the levels of secreted C3a and the expression of C3aR protein and mRNA in HCC cells. We further found that AA I-induced C3a/C3AR activation was involved in these effects. AA I-induced epithelial-to-mesenchymal transition (EMT), cell migration, and invasion were decreased by C3aR inhibition. Overall, our results suggest that AA I induces HCC cell migration and invasion through the EMT process, which is regulated by C3a/C3aR axis activation.

20(S)-ginsenoside Rh2 induces caspase-dependent promyelocytic leukemia-retinoic acid receptor A degradation in NB4 cells via Akt/Bax/caspase9 and TNF-α/caspase8 signaling cascades.

BACKGROUND: Acute promyelocytic leukemia (APL) is a hematopoietic malignancy driven by promyelocytic leukemia-retinoic acid receptor A (PML-RARA) fusion gene. The therapeutic drugs currently used to treat APL have adverse effects. 20(S)-ginsenoside Rh2 (GRh2) is an anticancer medicine with high effectiveness and low toxicity. However, the underlying anticancer mechanisms of GRh2-induced PML-RARA degradation and apoptosis in human APL cell line (NB4 cells) remain unclear. METHODS: Apoptosis-related indicators and PML-RARA expression were determined to investigate the effect of GRh2 on NB4 cells. Z-VAD-FMK, LY294002, and C 87, as inhibitors of caspase, and the phosphatidylinositol 3-kinase (PI3K) and tumor necrosis factor-α (TNF-α ) pathways were used to clarify the relationship between GRh2-induced apoptosis and PML-RARA degradation. RESULTS: GRh2 dose- and time-dependently decreased NB4 cell viability. GRh2-induced apoptosis, cell cycle arrest, and caspase3, caspase8, and caspase9 activation in NB4 cells after a 12-hour treatment. GRh2-induced apoptosis in NB4 cells was accompanied by massive production of reactive oxygen species, mitochondrial damage and upregulated Bax/Bcl-2 expression. GRh2 also induced PML/PML-RARA degradation, PML nuclear bodies formation, and activation of the downstream p53 pathway in NB4 cells. Z-VAD-FMK inhibited caspase activation and significantly reversed GRh2-induced apoptosis and PML-RARA degradation. GRh2 also upregulated TNF-α expression and inhibited Akt phosphorylation. LY294002, an inhibitor of the PI3K pathway, enhanced the antitumor effects of GRh2, and C 87, an inhibitor of the TNF-α pathway, reversed NB4 cell viability, and GRh2-mediated apoptosis in a caspase-8-dependent manner. CONCLUSION: GRh2 induced caspase-dependent PML-RARA degradation and apoptosis in NB4 cells via the Akt/Bax/caspase9 and TNF-α/caspase8 pathways.

Honokiol induces paraptosis-like cell death of acute promyelocytic leukemia via mTOR & MAPK signaling pathways activation.

Acute promyelocytic leukemia (APL) is a blood system disease caused by the accumulation of a large number of immature blood cells in bone marrow. Although the introduction of all-trans retinoic acid (ATRA) and arsenic has reached a high level of complete remission rate and 5-year disease-free survival rate, the occurrence of various adverse reactions still severely affects the quality of life of patients. As a natural product, honokiol (HNK) has the advantages of low toxicity and high efficiency, and it is a potential drug for the treatment of cancer. Since cancer cells can escape apoptotic cell death through multiple adaptive mechanisms, HNK, a drug that induces cancer cell death in a nonapoptotic way, has attracted much interest. We found that HNK reduced the viability of human APL cell line (NB4 cells) by inducing paraptosis-like cell death. The process was accompanied by excessive reactive oxygen species (ROS), mitochondrial damage, endoplasmic reticulum stress, and increased microtubule-associated protein 1 light chain 3 (LC3) processing. The inactivation of proteasome activity was the main cause of misfolded and unfolded protein accumulation in endoplasmic reticulum, such as LC3II/I and p62. This phenomenon could be alleviated by adding cycloheximide (CHX), a protein synthesis inhibitor. We found that mTOR signaling pathway participated in paraptosis-like cell death induced by HNK in an autophagy-independent process. Moreover, the mitogen-activated protein kinase (MAPK) signaling pathway induced paraptosis of NB4 cells by promoting endoplasmic reticulum stress. In summary, these findings indicate that paraptosis may be a new way to treat APL, and provide novel insights into the potential mechanism of paraptosis-like cell death.

Integration of transcriptomic, proteomic and metabolomic data to reveal the biological mechanisms of AAI injury in renal epithelial cells.

Overexposure to aristolochic acid I (AAI) can induce aristolochic acid nephropathy (AAN). However, the comprehensive mechanisms of AAI-induced nephrotoxicity have not been entirely explicated. To investigate the toxicological mechanisms by which AAI induces renal injury, human kidney cells (HK-2 cells) were subjected to comprehensive transcriptomic, proteomic and metabolomic analyses. The transcriptomic analysis identified a total of 7749 differentially expressed genes (DEGs) after AAI treatment, while the proteomic analysis found 598 differentially expressed proteins (DEPs) after AAI treatment. The metabolomic analysis revealed 49 and 42 differentially expressed metabolites (DEMs) in the positive and negative ion modes, respectively. Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed on these DEGs, DEPs and DEMs. The results of the comprehensive analyses of transcripts, proteins, and metabolites indicated that the DEGs, DEPs, and DEMs were jointly regulated in three ways. These genes, proteins and metabolites and their related dysregulated pathways may be promising targets for research on the mechanisms of AAI injury in human renal epithelial cells. This study provides large-scale omics data that can be used to formulate new strategies for the prevention, rapid diagnosis, and treatment of AAI injury.

Diosbulbin B-Induced Mitochondria-Dependent Apoptosis in L-02 Hepatocytes is Regulated by Reactive Oxygen Species-Mediated Autophagy.

Aim: Diosbulbin B (DB) is a major diterpenoid compound found in Dioscorea bulbifera L, a traditional medicinal herb in China. Clinical reports have confirmed that Dioscorea bulbifera L. can induce significant hepatotoxicity. In this study, we showed that DB can induce mitochondria-dependent apoptosis and investigated the role of autophagy in DB-induced hepatotoxicity in L-02 hepatocytes. Methods: L-02 hepatocytes were treated with different concentrations of DB for 48 h, after which indicators of autophagy and apoptosis were measured. 3-Methyladenine (3-MA) and rapamycin (Rapa) were used as inhibitor and agonist of autophagy, respectively. Furthermore, the reactive oxygen species (ROS) scavenger N-acetyl-l-cysteine (NAC) was used in combination with DB to evaluate the relationship between ROS and autophagy. Results: L-02 cell viability was significantly decreased after treatment with DB for 48 h. Additionally, DB induced concentration-dependent apoptosis and autophagy and increased the activities of caspase-3, caspase-9, alanine aminotransferase (ALT), and aspartate transaminase (AST), and induced excessive leakage of lactate dehydrogenase (LDH). Inhibition of autophagy by 3-MA increased DB-induced apoptosis, resulting in aggravation of hepatotoxicity. Conversely, treatment with Rapa increased malondialdehyde (MDA) content and reduced superoxide dismutase (SOD) activity. Moreover, we found that DB treatment increased the level of intracellular ROS, decreased the mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) production, and caused abnormal opening of the mitochondrial permeability transition pore (mPTP), which were finally restored by the ROS scavenger NAC. Conclusions: Accumulation of ROS can induce mitochondria-dependent apoptosis and likely to play a key role in DB-induced hepatocellular injury. Activation of autophagy may inhibit apoptosis, but also reduces antioxidant capacity.

Aristolochic acid I aggravates renal injury by activating the C3a/C3aR complement system.

Previous studies have reported that the complement system is unconventionally activated in many kinds of glomerulonephritis. Multiple complement components participate in the pathogenic process by triggering immune response or other intracellular signaling pathways. Here, we have investigated the role of C3a and its receptor C3aR in aristolochic acid nephropathy (AAN), which, is featured with progressive interstitial fibrosis. Over release of C3a and increased expression of C3aR parallels to the up-regulation of α-SMA and TGF-ß1 in AAN, which appeared to promote epithelial-mesenchymal-transition (EMT). To identify the role of complement activation in AAN, we used an inhibitor of C3aR (C3aRA) to block the coupling of C3a to its receptor. Our results confirmed from decreased EMT, the protective effect of C3aRA in cell apoptosis and inflammatory response induced by aristolochic acid I. These results showed that C3a and its receptor C3aR played pathogenic roles in AAN, and renal tubular epithelial cells were potentially pivotal targets of complement activation that could cause pro-fibrotic effects.