Effect of Pyridoxine Derivative B6NO on Transcription Factor Nrf2 Activity and Cytotoxic Properties of Doxorubicin In Vitro.

The effect of a new pyridoxine derivative B6NO on doxorubicin cytotoxicity and Nrf2-dependent cellular processes in vitro was studied. Antioxidant B6NO enhances the cytotoxic effect of doxorubicin on tumor cells, which is associated with G2/M cell division arrest and an increase in activity of proapoptotic enzyme caspase-3. The antioxidant promotes intracellular accumulation and nuclear translocation of Nrf2 transcription factor in non-tumor and tumor cells. In non-tumor cells, B6NO increases the expression of antioxidant system proteins and reduces ROS generation in the presence of doxorubicin. In tumor cells, no activation of Nrf2-dependent processes occurs under the action of the antioxidant. Our findings demonstrate the prospect of further studies of pyridoxine derivatives as antioxidants to reduce adverse reactions during chemotherapy.

CDK4/6 activity is required during G2 arrest to prevent stress-induced endoreplication.

Cell cycle events are coordinated by cyclin-dependent kinases (CDKs) to ensure robust cell division. CDK4/6 and CDK2 regulate the growth 1 (G1) to synthesis (S) phase transition of the cell cycle by responding to mitogen signaling, promoting E2F transcription and inhibition of the anaphase-promoting complex. We found that this mechanism was still required in G2-arrested cells to prevent cell cycle exit after the S phase. This mechanism revealed a role for CDK4/6 in maintaining the G2 state, challenging the notion that the cell cycle is irreversible and that cells do not require mitogens after passing the restriction point. Exit from G2 occurred during ribotoxic stress and was actively mediated by stress-activated protein kinases. Upon relief of stress, a significant fraction of cells underwent a second round of DNA replication that led to whole-genome doubling.

The bistable mitotic switch in fission yeast.

In favorable conditions, eukaryotic cells proceed irreversibly through the cell division cycle (G1-S-G2-M) in order to produce two daughter cells with the same number and identity of chromosomes of their progenitor. The integrity of this process is maintained by "checkpoints" that hold a cell at particular transition points of the cycle until all requisite events are completed. The crucial functions of these checkpoints seem to depend on irreversible bistability of the underlying checkpoint control systems. Bistability of cell cycle transitions has been confirmed experimentally in frog egg extracts, budding yeast cells and mammalian cells. For fission yeast cells, a recent paper by Patterson et al. (2021) provides experimental evidence for an abrupt transition from G2 phase into mitosis, and we show that these data are consistent with a stochastic model of a bistable switch governing the G2/M checkpoint. Interestingly, our model suggests that their experimental data could also be explained by a reversible/sigmoidal switch, and stochastic simulations confirm this supposition. We propose a simple modification of their experimental protocol that could provide convincing evidence for (or against) bistability of the G2/M transition in fission yeast.

AR-V7 expression facilitates accelerated G2/M phase transition in castration-resistant prostate cancer.

The emergence of AR-V7, a truncated isoform of AR upon androgen deprivation therapy treatment, leads to the development of castration resistant prostate cancer (CRPC). Understanding mechanisms that regulate AR-V7 expression is critical for developing newer therapeutic strategies. In this study, we have investigated the regulation of AR-V7 during cell cycle and identified a distinct pattern of periodic fluctuation, peaking during G2/M phase. This fluctuation correlates with the expression of Cdc-2 like kinase 1 (CLK1) and phosphorylated serine/arginine-rich splicing factor 1 (p-SRSF1) during these phases, pointing towards their role in AR-V7 generation. Functional assays reveal that CLK1 knockdown prolongs the S phase, leading to altered cell cycle distribution and increased accumulation of AR-V7 and pSRSF1 in G1/S phase. Conversely, CLK1 overexpression rescues AR-V7 and p-SRSF1 levels in the G2/M phase, consistent with observed cell cycle alterations upon AR-V7 knockdown and overexpression in CRPC cells. Furthermore, overexpression of kinase-deficient CLK1 mutant leads to diminished AR-V7 levels during G2/M, underlining the essential contribution of CLK1's kinase activity in modulating AR-V7 expression. Collectively, our findings, for the first time, show periodic regulation of AR-V7 expression, its effect on cell cycle progression and the critical role of CLK1-pSRSF1 axis in modulating AR-V7 expression throughout the cell cycle.

Investigation of prunetrin induced G2/M cell cycle arrest and apoptosis via Akt/mTOR/MAPK pathways in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) stands as a leading cause of mortality, and despite recent advancements in the overall survival rates, the prognosis remains dismal. Prunetin 4-O-glucoside (Prunetrin or PUR), an active compound derived from Prunus sp., was explored for its impact on HepG2 and Huh7 cells. The cytotoxicity assessment revealed a notable reduction in cell viability in both cell lines, while exhibiting non-toxicity towards HaCaT cells. Colony formation studies underscored PUR's inhibitory effect on cell proliferation, dose-dependently. Mechanistically, PUR downregulated cell cycle proteins (CDC25c, Cdk1/CDC2, and Cyclin B1), inducing G2/M phase arrest, corroborated by flow cytometry. Western blot analyses exhibited dose-dependent cleavages of PARP and caspase 3, indicative of apoptosis. Treatment with the apoptotic inhibitor z-vmd-fmk provided evidence of PUR-induced apoptosis. Annexin V and PI flow cytometry further affirmed apoptotic induction. Enhanced expression of cleaved-caspase 9 and the pro-apoptotic protein Bak, coupled with reduced anti-apoptotic Bcl-xL, and affirmed PUR's induction of intrinsic apoptosis. Additionally, PUR activated the MAPK pathway, evidenced by elevated phospho p38 and phospho ERK expressions in both cell lines. Notably, a concentration-dependent decrease in mTOR and Akt expressions indicated PUR's inhibition of the Akt/mTOR pathway in HepG2 and Huh7 cells. These findings illuminate PUR's multifaceted impact, revealing its potential as a promising therapeutic agent against HepG2 and Huh7 cells through modulation of cell cycle, apoptosis, and key signaling pathways.

Bisphenol A promotes cell death in healthy respiratory system cells through inhibition of cell proliferation and induction of G2/M cell cycle arrest.

Bisphenol A (BPA) is a substance that can harm the environment and human health by interfering with the normal functioning of the body's hormonal system. It is commonly found in various plastic-based products such as cosmetics, canned foods, beverage containers, and medical equipment and as well as it can also be absorbed by inhalation. There have been limited studies on the effects of BPA on lung fibroblasts, and it is still unclear how high levels of BPA can impact respiratory system cells, particularly the lungs and trachea. In this research, we aimed to investigate the cell cycle disruption potential of BPA on respiratory system cells by examining healthy trachea and lung cells together for the first time. The findings indicated that BPA exposure can alter the healthy cells' morphology, leading to reduced cellular viability that has been assessed by MTT and SRB assays. BPA treatment was able to activate caspase3 as expected, which could cause apoptosis in treated cells. Although the highest dose of BPA did not increase the apoptotic rate of rat trachea cells, it remarkably caused them to become necrotic (52.12%). In addition to quantifying the induction of apoptosis and necrosis by BPA, cell cycle profiles were also determined using flow cytometry. Thereby, BPA treatment unexpectedly inhibited the cell cycle's progression by causing G2/M cell cycle arrest in both lung and tracheal cells, which hindered cell proliferation. The findings of the study suggested that exposure to BPA could lead to serious respiratory problems, even respiratory tract cancers via alterations in the cell cycle.

Impaired TFEB-mediated autophagy-lysosome fusion promotes tubular cell cycle G2/M arrest and renal fibrosis by suppressing ATP6V0C expression and interacting with SNAREs.

Increasing evidence suggests that autophagy plays a major role during renal fibrosis. Transcription factor EB (TFEB) is a critical regulator of autophagy- and lysosome-related gene transcription. However, the pathophysiological roles of TFEB in renal fibrosis and fine-tuned mechanisms by which TFEB regulates fibrosis remain largely unknown. Here, we found that TFEB was downregulated in unilateral ureteral obstruction (UUO)-induced human and mouse fibrotic kidneys, and kidney-specific TFEB overexpression using recombinant AAV serotype 9 (rAAV9)-TFEB in UUO mice alleviated renal fibrosis pathogenesis. Mechanically, we found that TFEB's prevention of extracellular matrix (ECM) deposition depended on autophagic flux integrity and its subsequent blockade of G2/M arrest in tubular cells, rather than the autophagosome synthesis. In addition, we together RNA-seq with CUT&Tag analysis to determine the TFEB targeted gene ATP6V0C, and revealed that TFEB was directly bound to the ATP6V0C promoter only at specific site to promote its expression through CUT&Run-qPCR and luciferase reporter assay. Interestingly, TFEB induced autophagic flux integrity, mainly dependent on scaffold protein ATP6V0C-mediated autophagosome-lysosome fusion by bridging with STX17 and VAMP8 (major SNARE complex) by co-immunoprecipitation analysis, rather than its mediated lysosomal acidification and degradation function. Moreover, we further investigated the underlying mechanism behind the low expression of TEFB in UUO-induced renal fibrosis, and clearly revealed that TFEB suppression in fibrotic kidney was due to DNMT3a-associated TFEB promoter hypermethylation by utilizing methylation specific PCR (MSP) and bisulfite-sequencing PCR (BSP), which could be effectively recovered by 5-Aza-2'-deoxycytidine (5A-za) to alleviate renal fibrosis pathogenesis. These findings reveal for the first time that impaired TFEB-mediated autophagosome-lysosome fusion disorder, tubular cell G2/M arrest and renal fibrosis appear to be sequentially linked in UUO-induced renal fibrosis and suggest that DNMT3a/TFEB/ATP6V0C may serve as potential therapeutic targets to prevent renal fibrosis.

Telocinobufagin, a PLK1 suppressor that inhibits tumor growth and metastasis by modulating CDC25c and CTCF in HNSCC cells.

BACKGROUND: The high metastasis and mortality rates of head and neck squamous cell carcinoma (HNSCC) urgently require new treatment targets and drugs. A steroidal component of ChanSu, telocinobufagin (TBG), was verified to have anti-cancer effects in various tumors, but its activity and mechanism in anti-HNSCC were still unknown. PURPOSE: This study tried to demonstrate the anti-tumor effect of TBG on HNSCC and verify its potential mechanism. METHODS: The effect of TBG on cell proliferation and metastasis were performed and the TBG changed genes were detected by RNA-seq analysis in HNSCC cells. The GSEA and PPI analysis were used to identify the pathways targeted for TBG-regulated genes. Meanwhile, the mechanism of TBG on anti-proliferative and anti-metastasis were investigated in vitro and in vivo. RESULTS: The in vitro and in vivo experiments confirmed that TBG has favorable anti-tumor effects by induced G2/M phase arrest and suppressed metastasis in HNSCC cells. Further RNA-seq analysis demonstrated the genes regulated by TBG were enriched at the G2/M checkpoint and PLK1 signaling pathway. Then, the bioinformatic analysis of clinical data found that high expressed PLK1 were closely associated with poor overall survival in HNSCC patients. Furthermore, PLK1 directly and indirectly modulated G2/M phase and metastasis (by regulated CTCF) in HNSCC cells, simultaneously. TBG significantly inhibited the protein levels of PLK1 in both phosphorylated and non-phosphorylated forms and then, in one way, inactivated PLK1 failed to activate G2/M phase-related proteins (including CDK1, CDC25c, and cyclin B1). In another way, be inhibited PLK1 unable promote the nuclear translocation of CTCF and thus suppressed HNSC cell metastasis. In contrast, the anti-proliferative and anti-metastasis effects of TBG on HNSCC cell were vanished when cells high-expressed PLK1. CONCLUSION: The present study verified that PLK1 mediated TBG induced anti-tumor effect by modulated G2/M phase and metastasis in HNSCC cells.

Protective Mechanisms of Various Active Substances on Cell DNA Damage and Apoptosis Induced by Deoxynivalenol.

Deoxynivalenol (DON) is a secondary metabolite of fungi that is harmful to humans and animals. This study examined the protective effects of natural substances, including resveratrol, quercetin, vitamin E, vitamin C, and microbe-derived antioxidants (MA), on both human gastric mucosal cells (GES-1) and pig small intestinal epithelial cells (IPEC-1) when induced by DON. Cells were incubated with active substances for 3 h and then exposed to DON for 24 h. The oxidative stress index, cell cycle, and apoptosis were measured. As compared to cells treated only with DON, pretreatment with active substances improved the balance of the redox status in cells caused by DON. Specifically, quercetin, vitamin E, vitamin C, and MA showed the potential to alleviate the G2 phase cell cycle arrest effect that was induced by DON in both kinds of cells. It was observed that vitamin E and vitamin C can alleviate DON-induced apoptosis and the G2 phase cycle arrest effect mediated via the ATM-Chk 2-Cdc 25C and ATM-P53 signaling pathways in GES-1 cells. In IPEC-1 cells, vitamin C and MA can alleviate both DON-induced apoptosis and the G2 phase cycle arrest effect via the ATM-Chk 2-Cdc 25C signaling pathway. Different bioactive substances utilize different protective mechanisms against DON in interacting with different cells. The proper addition of vitamin E and vitamin C to food can neutralize the toxic effect of DON, while the addition of vitamin C and MA to animal feed can reduce the harm DON does to animals.

Capivasertib combines with docetaxel to enhance anti-tumour activity through inhibition of AKT-mediated survival mechanisms in prostate cancer.

BACKGROUND/OBJECTIVE: To explore the anti-tumour activity of combining AKT inhibition and docetaxel in PTEN protein null and WT prostate tumours. METHODS: Mechanisms associated with docetaxel capivasertib treatment activity in prostate cancer were examined using a panel of in vivo tumour models and cell lines. RESULTS: Combining docetaxel and capivasertib had increased activity in PTEN null and WT prostate tumour models in vivo. In vitro short-term docetaxel treatment caused cell cycle arrest in the majority of cells. However, a sub-population of docetaxel-persister cells did not undergo G2/M arrest but upregulated phosphorylation of PI3K/AKT pathway effectors GSK3ß, p70S6K, 4E-BP1, but to a lesser extent AKT. In vivo acute docetaxel treatment induced p70S6K and 4E-BP1 phosphorylation. Treating PTEN null and WT docetaxel-persister cells with capivasertib reduced PI3K/AKT pathway activation and cell cycle progression. In vitro and in vivo it reduced proliferation and increased apoptosis or DNA damage though effects were more marked in PTEN null cells. Docetaxel-persister cells were partly reliant on GSK3ß as a GSK3ß inhibitor AZD2858 reversed capivasertib-induced apoptosis and DNA damage. CONCLUSION: Capivasertib can enhance anti-tumour effects of docetaxel by targeting residual docetaxel-persister cells, independent of PTEN status, to induce apoptosis and DNA damage in part through GSK3ß.

Different Impacts of DNA-PK and mTOR Kinase Inhibitors in Combination with Ionizing Radiation on HNSCC and Normal Tissue Cells.

Despite substantial advancements in understanding the pathomechanisms of head and neck squamous cell carcinoma (HNSCC), effective therapy remains challenging. The application of kinase inhibitors (KIs) in HNSCC, specifically mTOR and DNA-PK inhibitors, can increase radiosensitivity and therefore presents a promising strategy when used simultaneously with ionizing radiation (IR) in cancer treatment. Our study focused on the selective DNA-PK-inhibitor AZD7648; the selective mTOR-inhibitor Sapanisertib; and CC-115, a dual inhibitor targeting both mTOR and DNA-PK. The impact of these KIs on HNSCC and normal tissue cells was assessed using various analytical methods including cell death studies, cell cycle analysis, real-time microscopy, colony-forming assays and immunohistochemical staining for γH2AX and downstream mTOR protein p-S6. We detected a strong inhibition of IR-induced DNA double-strand break (DSB) repair, particularly in AZD7648-treated HNSCC, whereas normal tissue cells repaired DNA DSB more efficiently. Additionally, AZD7648 + IR treatment showed a synergistic decline in cell proliferation and clonogenicity, along with an elevated G2/M arrest and cell death in the majority of HNSCC cell lines. CC-115 + IR treatment led to an elevation in G2/M arrest, increased cell death, and a synergistic reduction in cell proliferation, though the effect was notably lower compared to the AZD7648 + IR- treated group. Sapanisertib led to a high cellular toxicity in both HNSCC and normal tissue cells, even in non-irradiated cells. Regarding cell proliferation and the induction of apoptosis and necrosis, Sapanisertib + IR was beneficial only in HPV+ HNSCC. Overall, this study highlights the potential of AZD7648 as a radiosensitizing agent in advanced-stage HPV-positive and negative HNSCC, offering a promising therapeutic strategy. However, the dual mTOR/DNA-PK-I CC-115 did not provide a distinct advantage over the use of selective KIs in our investigations, suggesting limited benefits for its application in KI + IR therapy. Notably, the selective mTOR-inhibitor Sapanisertib was only beneficial in HPV+ HNSCC and should not be applied in HPV- cases.

Arenobufagin, isolated from Bufo viridis toad venom, inhibits A549 cells proliferation by inducing apoptosis and G2/M cell cycle arrest.

Lung cancer is a significant contributor to cancer morbidity and mortality globally. Arenobufagin, a compound derived from Bufo viridis toad venom, has demonstrated the ability to inhibit cell growth in various cancer cell lines. However, our understanding of the role and mechanism of arenobufagin in lung cancer remains incomplete, necessitating further researches to fully elucidate its action mechanism. In this study, we further explored the impact of arenobufagin on A549 cells. The results revealed that it exerted a potent cytotoxic effect on A549 cells by inhibiting cell colony formation, promoting cell apoptosis, increasing reactive oxygen species (ROS) levels, and arresting A549 cells in G2/M phase. Collectively, our findings suggested that arenobufagin may have potential as a future therapeutic for lung cancer treatment.

Myt1 kinase inhibitors - Insight into structural features, offering potential frameworks.

The cell cycle includes two checkpoint arrests allowing to repair of damaged DNA. Many cancer cell lines exhibit weak G1 checkpoint mechanisms relying significantly more on the G2 checkpoint than do healthy cells. Inhibition of Myt1 kinase (PKMYT1), a forgotten member of the Wee family, cyclin-dependent kinase 1 (Cdk1) inhibitory kinase, target for G2 checkpoint abrogation, whose inhibition forces cells into premature unchecked mitosis resulting in cell death, is a promising concept for anticancer therapy. There are not many inhibitors of this emerging, potentially clinically important kinase. Herein, the valuable insight into structural features and binding mechanisms of diaminopyrimidines, aminoquinolines, quinazolines, pyrido[2,3-d]pyrimidines, pyrazolo[3,4-d]pyrimidines, and pyrrolo[2,3-b]quinoxalines, as well as finally made a general scheme of fragmented structures of Myt1 inhibitors with the enzyme, offer potential frameworks useful for future directions, for further chemical optimizations, in the discovery and the design of novel effective structures, potential therapeutics.

Promotion of ROS-mediated apoptosis, G2/M arrest, and autophagy by naringenin in non-small cell lung cancer.

Background: As lung cancer is the leading cause of cancer death worldwide, the development of new medicines is a crucial endeavor. Naringenin, a flavanone derivative, possesses anti-cancer and anti-inflammatory properties and has been reported to have cytotoxic effects on various cancer cells. The current study investigated the underlying molecular mechanism by which naringenin induces cell death in lung cancer. Methods: The expression of apoptosis, cell cycle arrest, and autophagy markers in H1299 and A459 lung cancer cells was evaluated using a terminal deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL), Western blot, Annexin V/PI stain, PI stain, acridine orange staining, and transmission electron microscopy (TEM). Using fluorescence microscopy, DALGreen was used to observe the degradation of p62, a GFP-LC3 plasmid was used to evaluate puncta formation, and a pcDNA3-GFP-LC3-RFP-LC3ΔG plasmid was used to evaluate autophagy flux. Furthermore, the anti-cancer effect of naringenin was evaluated in a subcutaneous H1299 cell xenograft model. Results: Naringenin treatment of lung cancer cells (H1299 and A459) reduced cell viability and induced cell cycle arrest. Pretreatment of cells with ROS scavengers (N-acetylcysteine or catalase) suppressed the naringenin-induced cleavage of apoptotic protein and restored cyclin-dependent kinase activity. Naringenin also triggered autophagy by mediating ROS generation, thereby activating AMP-activated protein kinase (AMPK) signaling. ROS inhibition not only inhibited naringenin-induced autophagic puncta formation but also decreased the ratio of microtubule-associated proteins 1A/1B light chain 3 II (LC3II)/LC3I and activity of the AMPK signaling pathway. Furthermore, naringenin suppressed tumor growth and promoted apoptosis in the xenograft mouse model. Conclusion: This study demonstrated the potent anti-cancer effects of naringenin on lung cancer cells, thereby providing valuable insights for developing small-molecule drugs that can induce cell cycle arrest, apoptosis, and autophagic cell death.

Exploring the in vitro potential of royal jelly against glioblastoma and neuroblastoma: impact on cell proliferation, apoptosis, cell cycle, and the biomolecular content.

Neuroblastoma and glioblastoma are the most commonly seen nervous system tumors, and their treatment is challenging. Relatively safe and easy acquisition of nutraceutical natural products make them suitable candidates for anticancer research. Royal jelly (RJ), a superfood, has many biological and pharmacological activities. This study was conducted to, for the first time, elucidate its anticancer efficiency, even in high doses, on neuroblastoma and glioblastoma cell lines through cell viability, apoptosis, cell cycle and biomolecular content evaluation. We performed experiments with RJ concentrations in the range of 1.25-10 mg mL-1 for 48 h. Cell viability assays revealed a notable cytotoxic effect of RJ in a concentration-dependent manner. Treatment with a high dose of RJ significantly increased the apoptotic cell population of both cell lines. Furthermore, we observed G0-G1 phase arrest in neuroblastoma cells but G2-M arrest in glioblastoma cells. All these cellular changes are closely associated with the alterations of the macromolecular makeup of the cells, such as decreased saturated lipid, protein, DNA and RNA amounts, protein conformational changes, decreased protein phosphorylation and increased protein carbonylation. These cellular changes are associated with RJ triggered-ROS formation. The clear segregation between the control and the RJ-treated groups proved these changes, obtained from the unsupervised and supervised chemometric analysis. RJ has good anticancer activity against nervous system cancers and could be safely used with current treatment strategies.

Bisphenol A regulates bladder cells responses via control of G2/M-phase cell cycle, apoptotic signaling, MAPK pathway, and transcription factor-associated MMP modulation.

Bisphenol A (BPA), an exogenous endocrine-disrupting chemical, is widely used to produce polycarbonate plastics. The widely used BPA has been detected in human urine samples, raising public anxiety about the detrimental effects of BPA on the bladder. In this study, we explored regulatory mechanisms for the adverse effects of BPA in human bladder BdFC and T24 cells. BPA induced extrinsic and intrinsic apoptosis and G2/M cell cycle arrest caused by the ATM-CHK1/CHK2-CDC25c-CDC2 signaling, which ultimately inhibited the growth of human bladder cells. We also found that BPA decreased the binding activity of AP-1 and NF-κB transcription factors in human bladder cells, which inhibited migration and invasion through matrix metallopeptidase-2 and -9 inactivation. Phosphorylation of MAPKs was implicated with BPA-mediated detrimental effects in human bladder cells. Collectively, our results provide a novel explanation for the underlying molecular mechanisms that BPA induces cytotoxicity in human bladder cells.

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.

AZD1775 and anti-PD-1 antibody synergistically sensitize hepatoma to radiotherapy.

BACKGROUND: Radiation (IR)-induced DNA damage triggers cell cycle arrest and has a suppressive effect on the tumor microenvironment (TME). Wee1, a cell cycle regulator, can eliminate G2/M arrest by phosphorylating cyclin-dependent kinase 1 (CDK1). Meanwhile, programed death-1/programed death ligand-1 (PD-1/PDL-1) blockade is closely related to TME. This study aims to investigate the effects and mechanisms of Wee1 inhibitor AZD1775 and anti-PD-1 antibody (anti-PD-1 Ab) on radiosensitization of hepatoma. METHODS: The anti-tumor activity of AZD1775 and IR was determined by 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) assay on human and mouse hepatoma cells HepG2, Hepa1-6, and H22. The anti-hepatoma mechanism of AZD1775 and IR revealed by flow cytometry and Western blot in vitro . A hepatoma subcutaneous xenograft mice model was constructed on Balb/c mice, which were divided into control group, IR group, AZD1775 group, IR + AZD1775 group, IR + anti-PD-1 Ab group, and the IR + AZD1775 + anti-PD-1 Ab group. Cytotoxic CD8 + T cells in TME were analyzed by flow cytometry. RESULTS: Combining IR with AZD1775 synergistically reduced the viability of hepatoma cells in vitro . AZD1775 exhibited antitumor effects by decreasing CDK1 phosphorylation to reverse the IR-induced G2/M arrest and increasing IR-induced DNA damage. AZD1775 treatment also reduced the proportion of PD-1 + /CD8 + T cells in the spleen of hepatoma subcutaneous xenograft mice. Further studies revealed that AZD1775 and anti-PD-1 Ab could enhance the radiosensitivity of hepatoma by enhancing the levels of interferon γ (IFNγ) + or Ki67 + CD8 T cells and decreasing the levels of CD8 + Tregs cells in the tumor and spleen of the hepatoma mice model, indicating that the improvement of TME was manifested by increasing the cytotoxic factor IFNγ expression, enhancing CD8 + T cells proliferation, and weakening CD8 + T cells depletion. CONCLUSIONS: This work suggests that AZD1775 and anti-PD-1 Ab synergistically sensitize hepatoma to radiotherapy by enhancing IR-induced DNA damage and improving cytotoxic CD8 + T cells in TME.

Indigofera suffruticosa aerial parts extract induce G2/M arrest and ATR/CHK1 pathway in Jurkat cells.

BACKGROUND: Indigofera suffruticosa Mill. is used as a folk medicine for treating patients with leukemia, however very little is known regarding the molecular mechanism of its anti-leukemic activity and the chemical profile of the active extract. The present study aimed to reveal the molecular effect of I. suffruticosa aerial parts extract (ISAE) on leukemia cells and its chemical constituents. METHODS: Cytotoxicity of ISAE were determined by resazurin viability assay, multitox - Glo multiplex cytotoxicity assay, and Annexin V staining assay. Cell cycle profiles were revealed by propidium iodide staining assay. The effects of ISAE on G2/M arrest signaling and DNA damage were evaluated by Western blot assay and phospho-H2A.X staining assay. The chemical profile of ISAE were determined by tandem mass spectroscopy and molecular networking approach. RESULTS: We showed that the acute lymphoblastic leukemia cell line Jurkat cell was more responsive to ISAE treatment than other leukemia cell lines. In contrast, ISAE did not induce cytotoxic effects in normal fibroblast cells. Cell cycle analysis revealed that ISAE triggered G2/M arrest in Jurkat cells in dose- and time-dependent manners. Elevation of annexin V-stained cells and caspase 3/7 activity suggested ISAE-induced apoptosis. Furthermore, ISAE alone could increase the phosphorylation of CDK1 at Y15 and activate the ATR/CHK1/Wee1/CDC25C signaling pathway. However, the addition of caffeine, a widely used ATR inhibitor to ISAE, reduced the phosphorylation of ATR, CHK1, and CDK1, as well as G2/M arrest in Jurkat cells. Moreover, increased phospho-H2A.X stained cells indicated the involvement of DNA damage in the anti-leukemic effect of ISAE. Finally, qualitative analysis using UPLC-tandem mass spectroscopy and molecular networking revealed that tryptanthrin was the most abundant organoheterocyclic metabolite in ISAE. At equivalent concentrations to ISAE, tryptanthrin induced G2/M arrest of Jurkat cells, which can be prevented by caffeine. CONCLUSIONS: ISAE causes G2/M arrest via activating ATR/CHK1/CDK1 pathway and tryptanthrin is one of the active components of ISAE. Our findings provide subtle support to the traditional use of I. suffruitcosa in leukemia management in folk medicine.

Marinopyrrole derivative MP1 as a novel anti-cancer agent in group 3 MYC-amplified Medulloblastoma.

BACKGROUND: Medulloblastoma (MB) patients with MYC oncogene amplification or overexpression exhibit extremely poor prognoses and therapy resistance. However, MYC itself has been one of the most challenging targets for cancer treatment. Here, we identify a novel marinopyrrole natural derivative, MP1, that shows desirable anti-MYC and anti-cancer activities in MB. METHODS: In this study, using MYC-amplified (Group 3) and non-MYC amplified MB cell lines in vitro and in vivo, we evaluated anti-cancer efficacies and molecular mechanism(s) of MP1. RESULTS: MP1 significantly suppressed MB cell growth and sphere counts and induced G2 cell cycle arrest and apoptosis in a MYC-dependent manner. Mechanistically, MP1 strongly downregulated the expression of MYC protein. Our results with RNA-seq revealed that MP1 significantly modulated global gene expression and inhibited MYC-associated transcriptional targets including translation/mTOR targets. In addition, MP1 inhibited MYC-target metabolism, leading to declined energy levels. The combination of MP1 with an FDA-approved mTOR inhibitor temsirolimus synergistically inhibited MB cell growth/survival by downregulating the expression of MYC and mTOR signaling components. Our results further showed that as single agents, both MP1 and temsirolimus, were able to significantly inhibit tumor growth and MYC expression in subcutaneously or orthotopically MYC-amplified MB bearing mice. In combination, there were further anti-MB effects on the tumor growth and MYC expression in mice. CONCLUSION: These preclinical findings highlight the promise of marinopyrrole MP1 as a novel MYC inhibition approach for MYC-amplified MB.