Cytotoxicity of dioncophylline A and related naphthylisoquinolines in leukemia cells, mediated by NF-κB inhibition, angiogenesis suppression, G2/M cell cycle arrest, and autophagy induction.

BACKGROUND: Inhibition of NF-κB activity represents a strategy to treat acute myeloid leukemia, one of the most lethal leukemia types. Naphthylisoquinolines (NIQs) are cytotoxic alkaloids from lianas of the families Ancistrocladaceae and Dioncophyllaceae, which are indigenous to tropical rainforests. PURPOSE: Uncovering therapeutic possibilities and underlying molecular mechanisms of dioncophylline A and its derivatives towards NF-κB related cellular processes. METHODS: Resazurin-based cell viability assay was performed for dioncophylline A and three derivatives on wild-type CCRF-CEM and multidrug-resistant CEM/ADR5000 cells. Transcriptome analysis was executed to discover cellular functions and molecular networks associated with dioncophylline A treatment. Expression changes obtained by mRNA microarray hybridization were confirmed using qRT-PCR. Molecular docking was applied to predict the affinity of the NIQs with NF-κB. To validate the in silico approach, NF-κB reporter assays were conducted on HEK-Blue™ Null1 cells. Cell death mechanisms and cell cycle arrest were studied using flow cytometry. The potential activity on angiogenesis was evaluated with the endothelial cell tube formation assay on HUVECs using fluorescence microscopy. Intracellular NF-κB location in HEK-Blue™ Null1 cells was visualized with immunofluorescence. Finally, the anti-tumor activity of dioncophylline A was studied by a xenograft zebrafish model in vivo. RESULTS: Our study demonstrated that dioncophylline A and its derivatives exerted potent cytotoxicity on leukemia cells. Using Ingenuity Pathway Analysis, we identified the NF-κB network as the top network, and docking experiments predicted dioncophylline A and two of its derivatives sharing the same binding pocket with the positive control compound, triptolide. Dioncophylline A showed the best inhibitory activity in NF-κB reporter assays compared to its derivatives, caused autophagy rather than apoptosis, and induced G2/M arrest. It also prevented NF-κB translocation from the cytoplasm to the nucleus. Tube formation as an angiogenesis marker was significantly suppressed by dioncophylline A treatment. Finally, the remarkable anti-tumor activity of dioncophylline A was proven in zebrafish in vivo. CONCLUSION: Taken together, we report for the first time the molecular mechanism behind the cytotoxic effect of dioncophylline A on leukemia cells. Dioncophylline A showed strong cytotoxic activity, inhibited NF-κB translocation, significantly affected the NF-κB in silico and in vitro, subdued tube formation, induced autophagy, and exerted antitumor activity in vivo. Our findings enlighten both the cellular functions including the NF-κB signaling pathway and the cytotoxic mechanism affected by dioncophylline A.

Modes of Action of a Novel c-MYC Inhibiting 1,2,4-Oxadiazole Derivative in Leukemia and Breast Cancer Cells.

The c-MYC oncogene regulates multiple cellular activities and is a potent driver of many highly aggressive human cancers, such as leukemia and triple-negative breast cancer. The oxadiazole class of compounds has gained increasing interest for its anticancer activities. The aim of this study was to investigate the molecular modes of action of a 1,2,4-oxadiazole derivative (ZINC15675948) as a c-MYC inhibitor. ZINC15675948 displayed profound cytotoxicity at the nanomolar range in CCRF-CEM leukemia and MDA-MB-231-pcDNA3 breast cancer cells. Multidrug-resistant sublines thereof (i.e., CEM/ADR5000 and MDA-MB-231-BCRP) were moderately cross-resistant to this compound (<10-fold). Molecular docking and microscale thermophoresis revealed a strong binding of ZINC15675948 to c-MYC by interacting close to the c-MYC/MAX interface. A c-MYC reporter assay demonstrated that ZINC15675948 inhibited c-MYC activity. Western blotting and qRT-PCR showed that c-MYC expression was downregulated by ZINC15675948. Applying microarray hybridization and signaling pathway analyses, ZINC15675948 affected signaling routes downstream of c-MYC in both leukemia and breast cancer cells as demonstrated by the induction of DNA damage using single cell gel electrophoresis (alkaline comet assay) and induction of apoptosis using flow cytometry. ZINC15675948 also caused G2/M phase and S phase arrest in CCRF-CEM cells and MDA-MB-231-pcDNA3 cells, respectively, accompanied by the downregulation of CDK1 and p-CDK2 expression using western blotting. Autophagy induction was observed in CCRF-CEM cells but not MDA-MB-231-pcDNA3 cells. Furthermore, microarray-based mRNA expression profiling indicated that ZINC15675948 may target c-MYC-regulated ubiquitination, since the novel ubiquitin ligase (ELL2) was upregulated in the absence of c-MYC expression. We propose that ZINC15675948 is a promising natural product-derived compound targeting c-MYC in c-MYC-driven cancers through DNA damage, cell cycle arrest, and apoptosis.

Modulation of Intestinal Corticotropin-Releasing Hormone Signaling by the Herbal Preparation STW 5-II: Possible Mechanisms for Irritable Bowel Syndrome Management.

Corticotropin-releasing factor (CRF) mediates stress responses and alters the gut-brain axis, contributing to the pathogenesis of irritable bowel syndrome (IBS), which is recognized by abdominal pain accompanied by bowel habit disturbance. STW 5-II, a mixture of six herbal extracts, is clinically effective in functional dyspepsia and IBS. Here we aimed to establish an organoid-based stress-induced IBS-like model to investigate the mechanisms of action of STW 5-II. STW 5-II (10, 20, and 30 g/mL) was applied to intestinal organoids for 24 h before being treated with CRF (100 nM) for 48 h. The effects of STW 5-II on CRF signaling were investigated using several in vitro and in silico approaches. STW 5-II activities were further explored by in silico PyRx screening followed by molecular docking of the main 52 identified compounds in STW 5-II with both CRF receptors CRFR1 and CRFR2. CRF exposure stimulated inflammation and increased proinflammatory mediators, while STW 5-II dose-dependently counteracted these effects. STW 5-II inhibited CRF-induced claudin-2 overexpression and serotonin release. Docking of the STW 5-II constituents oleanolic acid and licorice saponin G2 to CRFR1 and CRFR2, respectively, showed a good affinity. These multi-target activities support and elucidate the clinically proven efficacy of STW 5-II in disorders of gut-brain interaction.

Pyrrolizidine alkaloids cause cell cycle and DNA damage repair defects as analyzed by transcriptomics in cytochrome P450 3A4-overexpressing HepG2 clone 9 cells.

Pyrrolizidine alkaloids (PAs) are a large group of highly toxic chemical compounds, which are found as cross-contaminants in numerous food products (e.g., honey), dietary supplements, herbal teas, and pharmaceutical herbal medicines. PA contaminations are responsible for serious hepatotoxicity and hepatocarcinogenesis. Health authorities have to set legal limit values to guarantee the safe consumption of plant-based nutritional and medical products without harmful health. Toxicological and chemical analytical methods are conventionally applied to determine legally permitted limit values for PAs. In the present investigation, we applied a highly sensitive transcriptomic approach to investigate the effect of low concentrations of five PAs (lasiocarpine, riddelliine, lycopsamine, echimidine, and monocrotaline) on human cytochrome P450 3A4-overexpressing HepG2 clone 9 hepatocytes. The transcriptomic profiling of deregulated gene expression indicated that the PAs disrupted important signaling pathways related to cell cycle regulation and DNA damage repair in the transfected hepatocytes, which may explain the carcinogenic PA effects. As PAs affected the expression of genes that involved in cell cycle regulation, we applied flow cytometric cell cycle analyses to verify the transcriptomic data. Interestingly, PA treatment led to an arrest in the S phase of the cell cycle, and this effect was more pronounced with more toxic PAs (i.e., lasiocarpine and riddelliine) than with the less toxic monocrotaline. Using immunofluorescence, high fractions of cells were detected with chromosome congression defects upon PA treatment, indicating mitotic failure. In conclusion, the tested PAs revealed threshold concentrations, above which crucial signaling pathways were deregulated resulting in cell damage and carcinogenesis. Cell cycle arrest and DNA damage repair point to the mutagenicity of PAs. The disturbance of chromosome congression is a novel mechanism of Pas, which may also contribute to PA-mediated carcinogenesis. Transcriptomic, cell cycle, and immunofluorescence analyses should supplement the standard techniques in toxicology to unravel the biological effects of PA exposure in liver cells as the primary target during metabolization of PAs.

Anti-inflammatory and tight junction protective activity of the herbal preparation STW 5-II on mouse intestinal organoids.

BACKGROUND: Irritable bowel syndrome (IBS) is a functional bowel disorder, in which recurrent abdominal pain is associated with defecation or a change in bowel habits. STW 5-II is a combination of six medicinal herbs with a clinically proven efficacy in managing IBS. AIM: This study aims to establish an in vitro IBS model using mouse intestinal organoids and to explore the anti-inflammatory and tight junction protective activities of the multi-herbal preparation STW 5-II. METHODS: Intestinal organoids were cultured in 1:1 Matrigel™ and medium domes. Inflammation and tight junction disruption were induced by a cocktail of cytokines (TNFα, IFNγ, IL-1ß, IL-6) and bacterial proteins (LPS, flagellin). Organoids were treated with different concentrations of STW 5-II, and its multi-target activity was assessed using microarray analyses, RT-qPCR, immunofluorescence, western blot, immunohistochemistry, and a FITC permeability assay. In addition, we analyzed the expression of pNF-κB, pSTAT1, iNOS and ZO-1. In silico analyses were conducted to predict and identify the active components that may be responsible in mediating the multi-target anti-inflammatory activity of STW 5-II. RESULTS: An organoid based IBS model was successfully established. STW 5-II effectively reduced the cytokines-induced overexpression of the pro-inflammatory mediators pNF-κB, pSTAT1 and iNOS. Moreover, STW 5-II attenuated cytokine-mediated downregulation of the tight junction protein, ZO-1. This finding was confirmed by a FITC permeability assay. In silico analyses revealed a promising inhibitory activity of some isolated compounds from STW 5-II against NF-κB, STAT1 and iNOS. CONCLUSION: STW 5-II possesses multiple anti-inflammatory as well as tight junction protective activities that could explain its clinically proven efficacy in managing IBS symptoms.

2α-Hydroxyalantolactone from Pulicaria undulata: activity against multidrug-resistant tumor cells and modes of action.

BACKGROUND: Sesquiterpene lactones having α-methylene-γ-lactone moiety are promising natural metabolites showing various biological activity. One of the major metabolites isolated from Pulicaria undulata, 2α-hydroxyalantolactone (PU-1), has not been investigated in detail yet. Multidrug resistance (MDR) represents a major obstacle for cancer chemotherapy and the capability of novel natural products to overcoming MDR is of great interest. PURPOSE: Exploring the molecular modes of action for potent natural product metabolites. METHODS: The resazurin reduction assay was employed to evaluate the cytotoxicity of PU-1 on sensitive and their corresponding drug-resistant cell lines (overexpressing P-glycoprotein, BCRP, ABCB5, ΔEGFR, or TP53 knockout). Gene expression profiling was performed by transcriptome-wide mRNA microarray in the human CCRF-CEM leukemic cells after treatment with PU-1. The top significantly up- or down-regulated genes were identified by Chipster program and analyzed using Ingenuity Pathway Analysis (IPA) software. Finally, flow cytometry and Western blotting were performed for cell cycle analyses and apoptosis detection. RESULTS: The sesquiterpene lactone, PU-1, showed potent cytotoxicity towards the drug-sensitive and -resistant cell lines. Transcriptome-wide mRNA expression profiling and pathway analysis pointed to genes involved in DNA damage response and G2/M cell cycle arrest. G2/M arrest was verified by flow cytometry and further confirmed by the upregulation of p21 and downregulation of p-CDC25C expression in Western blotting. Moreover, the suggested DNA damage checkpoint regulation was confirmed by immunofluorescence and Western blotting by upregulation of pS345 Chk1, p-H3 and γ-H2AX. Furthermore, PU-1 inhibited PI3K/AKT pathway, which is involved in signaling DNA damage and G2/M arrest. Cells ultimately induced apoptosis upon PU-1 treatment. CONCLUSIONS: PU-1 is a potent natural product inhibiting otherwise drug-resistant human tumor cell growth through DNA damage, G2/M cell cycle arrest and apoptosis.

Multiple modes of cell death in neuroendocrine tumors induced by artesunate.

BACKGROUND: The paucity of effective treatment in neuroendocrine tumors (NETs) encouraged us to investigate the therapeutic value of artesunate (ART) promised by its inhibitory effect against various tumors and broad safety profile. METHODS: We evaluated the impact of ART on three NET cell lines, BON-1, QGP-1 and NCI-H727 on cellular and molecular levels. RESULTS: Our results showed that ART induced endoplasmic reticulum (ER) stress through phosphorylation of eIF2α, which further gave rise to autophagy in all three NET cell lines. Specifically, apoptosis and ferroptosis were also observed in BON-1 cells, which made BON-1 cell line more vulnerable upon ART treatment. The different sensitivities presented on the three cell lines also associated with a differential regulation of p21 on the long run. Co-treatment with p21 inhibitor UC2288 showed an additive effect on QGP-1 and NCI-H727 cell lines indicating p21 upregulation in these two cell lines might confer resistance towards ART treatment. CONCLUSIONS: It is possible to include ART in the treatment of NETs in the future.

Adaptogens in chemobrain (Part II): Effect of plant extracts on chemotherapy-induced cytotoxicity in neuroglia cells.

BACKGROUND: Cancer chemotherapy-induced cognitive impairments are apparently associated with harmful effects on physiological functions of brain cells. Adaptogens, are known to exhibit neuroprotective effects and to increase cognitive functions in clinical studies. In our previous study (Seo et al., 2018), we demonstrated that selected adaptogenic extracts significantly attenuate cytostatic-induced regulation of more than 100 genes involved in the activation of neuronal death and inhibiting neurogenesis. Neuroprotective and cytoprotective activities of adaptogens rise the question about their possible impact on cytostatic effects of a chemotherapeutic combination of 5-fluorouracil, epirubicin and cyclophosphamide (FEC). AIM: The aim of this study was to assess the effects of selected adaptogenic herbal extracts, namely of andrographolide (AND), Herba Andrographidis (AP), Radix Eleutherococci (ES) genuine extracts, their fixed combination (AE), and the combination of three adaptogenic herbs, Rhodiola Radix, Shisandra Fructus and Eleutherococci Radix (RSE) on the cytotoxicity of a fixed combination of 5-fluorouracil, epirubicin and cyclophosphamide (FEC) on neuroglia cells. METHODS: Cytotoxicity of FEC, adaptogenic extracts and their combination with FEC was tested on isolated T98G cells in a wide range of concentrations of all tested compounds. RESULTS: FEC reproducibly inhibited the proliferation of T98G cells by 50% at concentrations of 5 × 10-1 µg/ml epirubicin, 500 × 10-1 µg/ml 5-fluorouracil and 20 × 10-1 µg/ml 4-hydroperoxycyclophosphamide after 24 h incubation of cells. These concentrations were subsequently used for experiments with adaptogenic extracts. The cytotoxic activity of FEC was not significantly changed in the presence of AND, ES and AE. Furthermore, it was potentiated by AP extract and RSE in concentrations of 0.06-6 µg/ml and 17.6-26.4 µg/ml. CONCLUSION: The neuroprotective effect of adaptogens did not attenuate the cytotoxic activity of FEC. Application of cytostatic drugs in combination with adaptogenic plant extracts likely have no impact in cytotoxic effect of FEC. Furthermore, AP and RSE potentiated the cytotoxic effects of FEC.

Adaptogens in chemobrain (Part III): Antitoxic effects of plant extracts towards cancer chemotherapy-induced toxicity - transcriptome-wide microarray analysis of neuroglia cells.

BACKGROUND: Toxicity of chemotherapeutics is a serious problem in cancer therapy. Adaptogens are known to increase adaptability and survival organisms. AIM: The aim of this study was to assess the effects of selected adaptogenic herbal extracts on FEC (fixed combination of 5-fluorouracil, epirubicin and cyclophosphamide) induced changes in transcriptome-wide microarray profiles of neuroglia cells. Another task of the study was to identify those genes, which are associated with FEC-induced hepato-, cardio- and nephrotoxicity to predict potential effects of andrographolide (AND), Andrographis herb, Eleutherococcus roots genuine extracts (ES), their fixed combination (AE) and the combination of Rhodiola roots, Schisandra berries and Eleutherococcus roots (RSE) on the organismal level. METHODS: Gene expression profiling was performed by transcriptome-wide mRNA microarray in the human T98G neuroglia cells after treatment with adaptogens. Interactive pathways downstream analysis was performed with data sets of significantly up- or down-regulated genes and predicted effects on cellular functions and diseases were identified by Ingenuity IPA database software. RESULT: Significant differences of transcriptome-wide microarray profiles were observed after treatment of T98G cells with FEC and after co-incubation with adaptogens. FEC induced deregulation of certain genes with suggested toxicity associated with liver fibroses, necrosis and congenital heart diseases. Co-incubation of AE with FEC prevented FEC-induced deregulation of 66 genes increasing organismal death, 37 genes decreasing cell survival, 37 genes decreasing DNA repair, 37 genes decreasing viral infection and some other functions, indicating on potential beneficial effects of AE. Furthermore, FEC-induced hepato-, nephro- and cardiotoxicity related to deregulation of genes was predictably attenuated by AE. Moreover, co-incubation of AE with FEC caused differential expression of genes, which presumably are beneficial for an organism during chemotherapy. They include predicted activation of DNA repair, activation of movement of antigen presenting cells and inhibition of muscle cells death. The main active constituent of AE is AND. Co-incubation of FEC only with AND results in deregulation of 10 genes causing death of breast cancer cells, decrease of liver toxicity and attenuation of organismal death. Co-incubation of ES extract with FEC showed that ES suppressed FEC-induced deregulation of genes, which inhibit organismal death and fertility. Co-incubation of FEC with RSE indicated potential hepatoprotective effect against FEC-induced apoptosis of liver cells presumably due to suppression of FEC-induced expressions of genes, which increased liver cell apoptosis. Simultaneously, RSE activated expression of genes inhibiting tumor growth. Though, microarray analysis did not provide final proof that the genes induced by the AE, AP and ES are responsible for the physiological effects observed in human patients following their oral administration, it provided insights into putative genes and directions for future research and possible implementation into practice. CONCLUSION: Application of cytostatic drugs in combination with adaptogenic plant extracts induced significant changes in transcriptome-wide microarray profiles of neuroglial cells. These changes indicate on potential beneficial effects of adaptogens on FEC induced adverse events in cancer chemotherapy.

Adaptogens in chemobrain (Part I): Plant extracts attenuate cancer chemotherapy-induced cognitive impairment - Transcriptome-wide microarray profiles of neuroglia cells.

BACKGROUND: Cancer chemotherapy-induced cognitive impairments are presumably associated with undesirable effects of chemotherapy on physiological functions of brain cells. Adaptogens are natural compounds or plant extracts increasing an organism's adaptability and survival in stress. They exhibited neuroprotective effects and increased cognitive functions in clinical studies in human beings. HYPOTHESIS: We hypothesized that selected adaptogenic plant extracts attenuate or prevent cancer chemotherapy-induced cognitive impairments. AIM: We assessed the effects of selected adaptogenic herbal extracts on FEC (fixed combination 5-fluorouracil, epirubicin and cyclophosphamide) induced changes in transcriptome-wide RNA microarray profiles of neuroglia cells. The aim of the study was to predict potential effects of andrographolide, Andrographis herb, Eleutherococcus root genuine extracts, their fixed combination (AE) and the combination of Rhodiola roots, Schisandra berries and Eleutherococcus roots (RSE) on cellular and physiological, mostly cognitive functions. METHODS: Gene expression profiling was performed by transcriptome-wide mRNA microarray in the human T98G neuroglia cells after treatment with adaptogens. Interactive pathways downstream analysis was performed with data sets of significantly up- or down-regulated genes and predicted effects on cellular functions and diseases were identified by Ingenuity IPA database software. RESULTS: FEC deregulated 67 genes involved in decrease of neuronal development, 37 genes involved in development of the sensory system, 12 genes in extension of axons, and 3 genes in migration of neurons. Co-incubation with Andrographis paniculata (AP) suppressed FEC-induced deregulation of a large number of genes involved in predicted activation of neuronal death and inhibition of neurogenesis, and 16 genes related to inhibition of several functions in the nervous system. Co-incubation with AE suppressed FEC-induced deregulation of a number of genes involved in predicted inhibition of axon extension, migration of T98G neuroglia cells, conduction of nerves and other genes related to regulations of some other functions in the nervous system. CONCLUSION: Application of cytostatic drugs in combination with apoptogenic plant extracts induced significant changes in transcriptome-wide mRNA microarray profiles of neuroglial cells. These changes indicate on potential beneficial effects on neuronal functions associated with mild cognitive impairments in cancer chemotherapy.

The Chinese herbal formula Free and Easy Wanderer ameliorates oxidative stress through KEAP1-NRF2/HO-1 pathway.

Posttraumatic stress disorder (PTSD) gains a lot of attention due to high prevalence and strong psychological upset, but the etiology remains undefined and effective treatment is quite limited. Growing studies demonstrated the involvement of oxidative stress in various psychiatry diseases, suggesting anti-oxidation therapy might be a strategy for PTSD treatment. Free and Easy Wanderer (FAEW) is a poly-herbal drug clinically used in China for hundreds of years in the treatment of psychiatric disorder. We hypothesized that FAEW exerts clinical effects through the activity against oxidative stress with fluoxetine as antidepressant control drug. Our results revealed that FAEW significantly reduced both endogenous and H2O2-induced exogenous ROS levels in the human glioblastoma T98G and neuroblastoma SH-SY5Y cell lines. Transcriptome-wide microarray analysis indicated NRF2/HO-1 as the common target of FAEW and fluoxetine. Western blotting assay proved that the two drugs promoted NRF2 release from KEAP1 in the cytoplasm and translocation to the nuclei in a KEAP1-dependent manner, the expression of the protein HO-1 increased accordingly, suggesting the participation of KEAP1-NRF2/HO-1 pathway. The chemical constituents of FAEW (i.e. paeoniflorin, baicalin) bound to KEAP1 in silico, which hence might be the effective substances of FAEW. In conclusion, FAEW counteracted H2O2-induced oxidative stress through KEAP1-NRF2/HO-1 pathway.

Cryptotanshinone deregulates unfolded protein response and eukaryotic initiation factor signaling in acute lymphoblastic leukemia cells.

BACKGROUND: Unfolded protein responses (UPR) determine cell fate and are recognized as anticancer targets. In a previous research, we reported that cryptotanshinone (CPT) exerted cytotoxic effects toward acute lymphoblastic leukemia cells through mitochondria-mediated apoptosis. PURPOSE: In the present study, we further investigated the role of UPR in CPT-induced cytotoxicity on acute lymphoblastic leukemia cells by applying tools of pharmacogenomics and bioinformatics. METHODS: Gene expression profiling was performed by mRNA microarray hybridization. Potential transcription factor binding motifs were identified in the promoter regions of the deregulated genes by Cistrome software. Molecular docking on eIF-4A and PI3K was performed to investigate the inhibitory activity of CPT on translation initiation. RESULTS: CPT regulated genes related to UPR and eIF2 signaling pathways. The DNA-Damage-Inducible Transcript 3 (DDIT3) gene, which is activated as consequence of UPR malfunction during apoptosis, was induced and validated by in vitro experiments. Transcription factor binding motif analysis of the microarrary-retrieved deregulated genes in the promoter region emphasized the relevance of transcription factors, such as ATF2, ATF4 and XBP1, regulating UPR and cell apoptosis. Molecular docking suggested inhibitory effects of CPT by binding to eIF-4A and PI3K providing evidence for a role of CPT's in the disruption of protein synthesis. CONCLUSION: CPT triggered UPR and inhibited protein synthesis via eIF-mediated translation initiation, potentially supporting CPT-induced cytotoxic effects toward acute leukemia cells.

Molecular mechanisms of rosmarinic acid from Salvia miltiorrhiza in acute lymphoblastic leukemia cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Rosmarinic acid (RA), a major hydrosoluble bioactive compound found in the Chinese medicinal herb, Salvia miltiorrhiza Bunge, which has been used in traditional Chinese medicine to treat various diseases, including cancer. However, the mechanisms have not been fully elucidated. AIM OF THE STUDY: Guided by microarray hybridization and Ingenuity Pathway Analysis, we identified modes of action of rosmarinic acid (RA) isolated from S. miltiorrhiza on acute lymphoblastic leukemia cells. MATERIALS AND METHODS: Microarray data were verified by independent methods: Real-time RT-PCR (mRNA expression), resazurin assay (cytotoxicity of RA towards parental CCRF-CEM, multidrug-resistant CEM/ADR5000 cells and normal lymphocytes), flow cytometry (cell cycle arrest, apoptosis, necroptosis, generation of reactive oxygen species (ROS), disruption of mitochondrial membrane potential (MMP)), single cell gel electrophoresis (DNA damage), molecular docking and gene promoter binding motif analysis (NFκB), Western blotting (nuclear NFκB translocation, PARP cleavage, caspase 3/7/9 expression), and fibronectin-based cell adhesion assay. RESULTS: RA dose-dependently inhibited CCRF-CEM and CEM/ADR5000 cells, but caused less cytotoxicity towards normal lymphocytes. RA simultaneously induced apoptosis and necrosis, as shown by cell morphology and annexin V-PI assay. DNA damage was dose-dependently induced without ROS generation, which subsequently led to cell cycle arrest. RA-stimulated MMP dysfunction activated PARP-cleavage and caspase-independent apoptosis. In accordance with molecular docking and gene promoter binding motif analyses, p65 translocation from the cytosol to the nucleus was blocked by RA, indicating a mechanistic role of the NFκB pathway to explain RA's action. RA affected cellular movement as evaluated by ameliorating cell adhesion to fibronectin. CONCLUSIONS: RA induced apoptosis and necrosis in a ROS-independent DNA damage and caspase-independent manner. These results may contribute to the rationale use of S. miltiorrhiza and RA in traditional medicine of leukemia.