G1 Arrest Research Articles

62 (PB052) - p53 mRNA rescue of tumor suppressor function prevents tumor growth and restores PARPi sensitivity in p53-deficient cancers in vitro and in vivo

Background: Loss of function in tumor suppressor genes is commonly associated with the onset/progression of cancer and the development of therapeutic resistance. The p53 tumor suppressor mutations resulting in loss of function are common across variety of tumors and control tumor cell proliferation in more than 50% of cancers. P53 null mutations are present in about 10% of p53 driven cancers. However, to date there is no effective treatment for rescuing p53 function in cancers. We have developed a new potent strategy combing p53 mRNA with a tumor selective nanocarrier, to rescue p53 tumor suppressor function as potential therapeutic approach in cancers. Methods: ADGN-technology is based on short amphipathic peptides that form stable neutral nanoparticles with mRNA. ADGN-531 nanoparticles containing full length p53-mRNA were evaluated on 20 different cancer cell lines (osteosarcoma, pancreas, colorectal, ovarian, lung, breast and prostate cancers) harboring different types of p53 mutations (null, deletion, nonsense, missense). Cell proliferation, cell cycle level and apoptosis activation were determined by flow cytometry, Elisa and Tunel assay. In-vivo efficacy of IV-administered ADGN-531 nanoparticles (0.5 mg/kg) was evaluated on colorectal SW403 (p53 deleted) and osteosarcoma SaOs2 (p53 null) mouse xenografts. Sensitivity to Veliparib (PARPi) following ADGN-531 treatment in vitro was evaluated on PARPI resistant SUM-149PT and OVCAR-8 cells and on PARPi sensitive MDA-MB436 cells. Results: ADGN-531 NPs markedly delay the growth of a large panel of cancer cells harboring p53-null or nonsense mutations, by inducing cell cycle arrest in G1 due to p21 upregulation and apoptosis following PUMA activation. We demonstrated that ADGN-531 mediated p53 function rescue is directly correlated to the type and level of p53 mutation in the cancer cells. ADGN-531 induces 60% to 80% cell proliferation inhibition in p53 null or nonsense mutated cells and 20 to 50% in p53 missense mutated cells. Intravenous-administration ADGN-531 containing wild type p53-mRNA (0.5 mg/kg) resulted in tumor growth inhibition of 90% and 70% in SaOs2 and SW403 xenografted mouse model, respectively. ADGN-531 NPs treatments are well tolerated, without inducing clinical toxicity or inflammatory response. We demonstrated that ADGN-531 mediated p53 rescue markedly improves (200 fold) and restores the sensitivity of ovarian and breast cancer cells to the PARPi veliparib. Conclusions: ADGN-531 nanoparticles are effective in rescuing P53 functions both in vitro and in vivo. Our study provides a proof-of-concept that restoration of tumor suppressor function by the ADGN-531 NP targeted delivery strategy could be combined together with other therapies for potent combinatorial cancer treatment. No conflict of interest.

Escape from G1 arrest during acute MEK inhibition drives the acquisition of drug resistance.

Mutations and gene amplifications that confer drug resistance emerge frequently during chemotherapy, but their mechanism and timing are poorly understood. Here, we investigate BRAFV600E amplification events that underlie resistance to the MEK inhibitor selumetinib (AZD6244/ARRY-142886) in COLO205 cells, a well-characterized model for reproducible emergence of drug resistance, and show that BRAF amplifications acquired de novo are the primary cause of resistance. Selumetinib causes long-term G1 arrest accompanied by reduced expression of DNA replication and repair genes, but cells stochastically re-enter the cell cycle during treatment despite continued repression of pERK1/2. Most DNA replication and repair genes are re-expressed as cells enter S and G2; however, mRNAs encoding a subset of factors important for error-free replication and chromosome segregation, including TIPIN, PLK2 and PLK3, remain at low abundance. This suggests that DNA replication following escape from G1 arrest in drug is more error prone and provides a potential explanation for the DNA damage observed under long-term RAF–MEK–ERK1/2 pathway inhibition. To test the hypothesis that escape from G1 arrest in drug promotes de novo BRAF amplification, we exploited the combination of palbociclib and selumetinib. Combined treatment with selumetinib and a dose of palbociclib sufficient to reinforce G1 arrest in selumetinib-sensitive cells, but not to impair proliferation of resistant cells, delays the emergence of resistant colonies, meaning that escape from G1 arrest is critical in the formation of resistant clones. Our findings demonstrate that acquisition of MEK inhibitor resistance often occurs through de novo gene amplification and can be suppressed by impeding cell cycle entry in drug.

Nuclear Beclin 1 Destabilizes Retinoblastoma Protein to Promote Cell Cycle Progression and Colorectal Cancer Growth.

Simple SummaryThe role of autophagy core-protein Beclin 1 in colorectal cancer (CRC) development remains controversial. Here, we show that nuclear Beclin 1 is up-regulated in CRC with a negative correlation to RB protein expression. Silencing of BECN1 up-regulates RB expression resulting in cell cycle G1 arrest and inhibition of xenograft tumor growth independent of p53. Ablation of BECN1 facilitates MDM2–MDMX complex formation to promote MDMX polyubiquitination and degradation, consequently leading to RB protein stabilization. These results reveal that nuclear Beclin 1 can promote CRC growth through modulation of RB protein stability and imply that nuclear Beclin 1 may be a prognostic indicator in human colorectal cancer.Autophagy is elevated in colorectal cancer (CRC) and is generally associated with poor prognosis. However, the role of autophagy core-protein Beclin 1 remains controversial in CRC development. Here, we show that the expression of nuclear Beclin 1 protein is upregulated in CRC with a negative correlation to retinoblastoma (RB) protein expression. Silencing of BECN1 upregulates RB resulting in cell cycle G1 arrest and growth inhibition of CRC cells independent of p53. Furthermore, ablation of BECN1 inhibits xenograft tumor growth through elevated RB expression and reduced autophagy, while simultaneous silencing of RB1 restores tumor growth but has little effect on autophagy. Mechanistically, knockdown of BECN1 promotes the complex formation of MDM2 and MDMX, resulting in MDM2-dependent MDMX instability and RB stabilization. Our results demonstrate that nuclear Beclin 1 can promote cell cycle progression through modulation of the MDM2/X-RB pathway and suggest that Beclin 1 promotes CRC development by facilitating both cell cycle progression and autophagy.

Suppression of intrahepatic cholangiocarcinoma cell growth by SKI via upregulation of the CDK inhibitor p21.

Cholangiocarcinoma (CC) has a poor prognosis and different driver genes depending on the site of onset. Intrahepatic CC is the second-most common liver cancer after hepatocellular carcinoma, and novel therapeutic targets are urgently needed. The present study was conducted to identify novel therapeutic targets by exploring differentially regulated genes in human CC. MicroRNA (miRNA) and mRNA microarrays were performed using tissue and serum samples obtained from 24 surgically resected hepatobiliary tumor cases, including 10 CC cases. We conducted principal component analysis to identify differentially expressed miRNA, leading to the identification of miRNA-3648 as a differentially expressed miRNA. We used an in silico screening approach to identify its target mRNA, the tumor suppressor Sloan Kettering Institute (SKI). SKI protein expression was decreased in human CC cells overexpressing miRNA-3648, endogenous SKI protein expression was decreased in human CC tumor tissues, and endogenous SKI mRNA expression was suppressed in human CC cells characterized by rapid growth. SKI-overexpressing OZ cells (human intrahepatic CC cells) showed upregulation of cyclin-dependent kinase inhibitor p21 mRNA and protein expression and suppressed cell proliferation. Nuclear expression of CDT1 (chromatin licensing and DNA replication factor 1), which is required for the G1/S transition, was suppressed in SKI-overexpressing OZ cells. SKI knockdown resulted in the opposite effects. Transgenic p21-luciferase was activated in SKI-overexpressing OZ cells. These data indicate SKI involvement in p21 transcription and that SKI-p21 signaling causes cell cycle arrest in G1, suppressing intrahepatic CC cell growth. Therefore, SKI may be a potential therapeutic target for intrahepatic CC.

EZH2 Regulates ANXA6 Expression via H3K27me3 and Is Involved in Angiotensin II-Induced Vascular Smooth Muscle Cell Senescence.

Objectives Abdominal aortic aneurysm (AAA) has a high risk of rupture of the aorta and is one of the leading causes of death in older adults. This study is aimed at confirming the influence and mechanism of the abnormally expressed ANXA6 gene in AAA. Methods Clinical samples were collected for proteome sequencing to screen for differentially expressed proteins. An Ang II-induced vascular smooth muscle cell (VSMC) aging model as well as an AAA animal model was used. Using RT-qPCR to detect the mRNA levels of EZH2, ANXA6, IK-6, and IL-8 in cells and tissues were assessed. Western blotting and immunohistochemistry staining were used apply for the expression of associated proteins in cells and tissues. SA-β-gal staining, flow cytometry, and DHE staining were used to detect senescent cells and the level of ROS. The cell cycle was assessed by flow cytometry. Arterial pathology was observed by HE staining. The aging of VSMCs in arterial tissue was assessed by coimmunofluorescence for α-SMA and p53. Results There were 24 differentially expressed proteins in the AAA clinical samples, including 10 upregulated protein and 14 downregulated protein, and the differential expression of ANXA6 was associated with vascular disease. Our study found that ANXA6 was highly expressed and EZH2 was lowly expressed in an Ang II-induced VSMC aging model. Knockdown of ANXA6 or overexpression of EZH2 inhibited Ang II-induced ROS, inhibited cell senescence, decreased Ang II evoked G1 arrest, and increased cells in G2 phase, while overexpression of ANXA6 played the opposite role. Overexpression of EZH2 inhibited ANXA6 expression by increasing H3K27me3 modification at the ANXA6 promoter. Simultaneous overexpression of EZH2 and the protective effect of EZH2 on cell senescence were partially reversed by ANXA6. Similarly, ANXA6 was highly expressed and EZH2 was lowly expressed in an Ang II-induced AAA animal model. Knockdown of ANXA6 and overexpression of EZH2 alleviated Ang II-induced VSMC senescence and inhibited AAA progression, while simultaneous overexpression of EZH2 and ANXA6 partially reversed the protective effect of EZH2 on AAA. Conclusion EZH2 regulates the ANXA6 promoter H3K27me3 modification, inhibits ANXA6 expression, alleviates Ang II-induced VSMC senescence, and inhibits AAA progression.

In Vivo Metabolites of Panaxadiol Inhibit HepG-2 Cell Proliferation by Inducing G1 Arrest and ROS-Mediated Apoptosis

In this study, 10 metabolites were obtained by collecting and extracting fecal samples after oral administration of panaxadiol (PD). Of these 10 metabolites, M7 (3β,21β,22α-hydroxy-24-norolean-12-ene), M8 (21β,22α-hydroxy-24-norolean-12-ene-3-one), M9 (3β,30α-hydroxy-24-norolean-22,30-epoxy-12-ene), and M10 (3β,21β-hydroxy-24-norolean-12-ene) were new compounds. MTT screening of the isolated compounds revealed that the inhibitions of cancer cells by M2, M4, M7, M8, and M10 were significantly stronger than that by the mother drug M0, with the activity of M2 being the most significant. Further, we investigated the anticancer mechanism of M2. The results showed that M2 significantly increased the level of ROS in cells; regulated the expressions of Bax, Bcl-2, and Cyt-C through the mitochondrial pathway; triggered the caspase cascade; and induced apoptosis. M2 could also induce G1 phase arrest and significantly regulate cell cycle-related proteins. In conclusion, the experimental results provide data for further study on the metabolic mechanism of PD in vivo and the potential of developing new anti-cancer drugs.

Abstract A02: Probing the roles of SUMOylation in B-cell lymphoma cells by using a selective SUMO inhibitor subasumstat (TAK-981)

Abstract SUMOylation is a post-translational modification that leads to covalent attachment of small ubiquitin-like modifiers (SUMO) proteins to a target protein. SUMOylation is essential for the regulation of genomic integrity, gene expression and intracellular signaling. Many tumors exhibit deregulation of SUMOylation pathway. TAK-981, a novel selective inhibitor of SUMO-activating enzyme (SAEi) has entered early phase II clinical trials. Here we studied SAEi in pre-clinical models of non-Hodgkin lymphoma (NHL). TAK-981 was provided by Takeda (Lexington, MA). Diffuse large B-cell lymphoma (DLBCL) cell lines and primary mantle cell lymphoma (MCL) cells were assayed for survival, mitochondrial function and metabolic phenotype. In vivo experiments were performed using DLBCL cell line xenografts and MCL PDX models. Treatment with TAK-981 restricted growth of DLBCL lines and led to rapid protein de-SUMOylation in a dose-dependent manner. TAK-981 induced pronounced DNA break in DLBCL cells (comet assay) and cell cycle arrest (propidium iodide). RNA-Seq and ATAC-Seq analysis of TAK-981-treated OCI-LY3 and U-2932 cells revealed upregulation of the NFκB, and OXPHOS signaling pathways. We noted that mitochondrial proteins were heavily de-SUMOylated upon treatment of NHL cells with TAK-981. In vitro exposure to TAK-981 resulted in mitochondrial membrane depolarization, loss of mitochondrial integrity (EM) and a change of mitochondrial dynamics (ICC for p-Drp1), accompanied by rapid accumulation of reactive oxygen species. Seahorse respirometry revealed dramatic downmodulation of OXPHOS. Metabolomic profiling of DLBCL cells treated with TAK981 exhibited downmodulation of TCA substrates. Exposure of primary MCL cells to TAK-981 in stromal conditions (CD40L- or BAFF expressing stroma) induced apoptosis and reduced OXPHOS. Next, we conducted genome-wide CRISPR-Cas9 loss-of-function library screens and identified that loss of genes in the NFκB, TP53 and DNA damage pathways contributed to resistance to SAEi in DLBCL cells. By means of genetic knockout we demonstrated that TP53 and its transcriptional target BAX contributed to TAK-981-induced G2 arrest and apoptosis, further implicating mitochondrial dysfunction in sensitivity to SAEi. For in vivo experiments, OCI-LY3 and U-2932 cells were inoculated subcutaneously in NSG mice. Once tumors reached 100 mm3, mice received twice-weekly IV doses of 7.5 mg/kg TAK-981 (over 5 weeks), leading to resolution of flank tumors and improved survival compared with control. Finally, we used an MCL PDX model where MCL cells were inoculated intravenously. Once cells became detectable in the blood, mice were treated as above. Treatment with TAK-981 resulted in delayed tumor expansion in blood and spleen and extended animal survival. TAK-981 exposed splenocytes exhibited a reduction of maximal respiration by Seahorse. In summary, pharmacologic SAEi with TAK-981 demonstrated pre-clinical activity in NHL models in vitro and in vivo, accompanied by DNA damage, mitochondrial dysfunction and metabolic reprogramming. Citation Format: Tingting Liu, Vi Lam, Duanchen Sun, Elana Thieme, Tamilla Nechiporuk, Daniel Bottomly, Olga V Danilova, Nur Bruss, Martina Cusan, Lili Wang, Shannon K McWeeney, Jeffrey W Tyner, Steve Kurtz, Zheng Xia, Alexey Danilov. Probing the roles of SUMOylation in B-cell lymphoma cells by using a selective SUMO inhibitor subasumstat (TAK-981) [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A02.

Abstract P054: Loss Of The Smooth-muscle-cell-angiotensin Ii-sensitive Lncrna Leads To Smc Hypertrophic And Hypertensive Remodeling Due To Cell Cycle Dysregulation.

Vascular smooth muscle cells (SMC) responses to increased levels of Ang II during hypertensive remodeling included enhanced vasoconstriction and hypertrophy due to the enlargement of the SMC on the vessel wall. Hypertrophic SMC usually present defects in cell division and cell polyploidy. However, the molecular mechanisms controlling SMC hypertrophy and polyploidy vs. hyperplasia are still not fully understood. Long-non-coding-RNAs (LncRNAs) are epigenetic regulators of gene expression influencing biological processes, including cell division fidelity. We discovered a novel lncRNA decreased in dedifferentiated SMC, the SMC-Ang II-Sensitive (SAS) lncRNA, which expression is reduced in response to Ang-II in cultured SMC and the aorta of hypertensive mice, suggesting a role in mediating hypertension-induced SMC hypertrophy. Publicly available transcriptional datasets revealed that SAS is preferentially expressed in SMC-rich tissues, including the aorta and renal artery, in humans and mice. Yet, the functional relevance of SAS in SMC has never been investigated. Knockdown of SAS reduces proliferation and migration in SMC treated with Platelet-Derived Growth Factor (PDGF-BB). SAS knockdown was also associated with distinct SMC hypertrophic morphological changes, including enlargement in cell size and polynucleation in vitro. We have generated a SAS KO mouse, and aortas from these mice present a higher number of polynucleated medial SMC than their WT littermates. These data correlate with a cell cycle arrest in G1 and senescence phenotype that SAS deficient SMC present due to dysregulation in cyclins expression. Furthermore, SAS defective cells show mitochondria hyperfusion and increased oxygen consumption that correlates with the observed senescence and arrest on the G1/S checkpoint, and it is exacerbated by treatment with Ang-II. Interestingly, treatment with Losartan, an Ang-II receptor inhibitor, rescues SAS expression on Ang-II treated SMC and diminishes SMC hypertrophy. Together, these observations suggest that a decrease in SAS causes SMC hypertrophy due to defects in cell cycle completion. SAS is a potent regulator of SMC morphology and is required for proper cell division and mitochondria organization.

RPRM deletion preserves hematopoietic regeneration by promoting EGFR-dependent DNA repair and hematopoietic stem cell proliferation post ionizing radiation.

Reprimo (RPRM), a target gene of p53, is a known tumor suppressor. DNA damage induces RPRM, which triggers p53-dependent G2 arrest by inhibiting cyclin B1/Cdc2 complex activation and promotes DNA damage-induced apoptosis. RPRM negatively regulates ataxia-telangiectasia mutated by promoting its nuclear-cytoplasmic translocation and degradation, thus inhibiting DNA damage. Therefore, RPRM plays a crucial role in DNA damage response. Moreover, the loss of RPRM confers radioresistance in mice, which enables longer survival and less severe intestinal injury after radiation exposure. However, the role of RPRM in radiation-induced hematopoietic system injury remains unknown. Herein, utilizing a RPRM-knockout mouse model, we found that RPRM deletion did not affect steady-state hematopoiesis in mice. However, RPRM knockout significantly alleviated radiation-induced hematopoietic system injury and preserved mouse hematopoietic regeneration in hematopoietic stem cells (HSCs) against radiation-induced DNA damage. Further mechanistic studies showed that RPRM loss significantly increased EGFR expression and phosphorylation in HSCs to activate STAT3 and DNA-PKcs, thus promoting HSC DNA repair and proliferation. These findings reveal the critical role of RPRM in radiation-induced hematopoietic system injury, confirming our hypothesis that RPRM may serve as a novel target for radiation protection.

Ivermectin and gemcitabine combination treatment induces apoptosis of pancreatic cancer cells via mitochondrial dysfunction

Pancreatic cancer is an aggressive cancer characterized by high mortality and poor prognosis, with a survival rate of less than 5 years in advanced stages. Ivermectin, an antiparasitic drug, exerts antitumor effects in various cancer types. This is the first study to evaluate the anticancer effects of the combination of ivermectin and gemcitabine in pancreatic cancer. We found that the ivermectin–gemcitabine combination treatment suppressed pancreatic cancer more effectively than gemcitabine alone treatment. The ivermectin–gemcitabine combination inhibited cell proliferation via G1 arrest of the cell cycle, as evidenced by the downregulation of cyclin D1 expression and the mammalian target of rapamycin (mTOR)/signal transducer and activator of transcription 3 (STAT-3) signaling pathway. Ivermectin–gemcitabine increased cell apoptosis by inducing mitochondrial dysfunction via the overproduction of reactive oxygen species and decreased the mitochondrial membrane potential. This combination treatment also decreased the oxygen consumption rate and inhibited mitophagy, which is important for cancer cell death. Moreover, in vivo experiments confirmed that the ivermectin–gemcitabine group had significantly suppressed tumor growth compared to the gemcitabine alone group. These results indicate that ivermectin exerts synergistic effects with gemcitabine, preventing pancreatic cancer progression, and could be a potential antitumor drug for the treatment of pancreatic cancer.

Diselenide-derivative of 3-pyridinol targets redox enzymes leading to cell cycle deregulation and apoptosis in A549cells.

The aim of present study was to understand the mechanism of action of 2,2'-diselenobis(3-pyridinol) or DISPOL in human lung cancer (A549) cells. A549 cells were treated with 10µM (∼IC50) of DISPOL for varying time points to corelate the intracellular redox changes with its cytotoxic effect. The results indicated that DISPOL treatment led to a time dependant decrease in the basal level of reactive oxygen species (ROS). Additionally, DISPOL treatment elevated the ratio of reduced (GSH) and oxidised (GSSG) glutathione by upregulating gamma-glutamylcysteine ligase (γ-GCL) involved in GSH biosynthesis and inhibiting the activities of redox enzymes responsible for GSH utilization and recycling, such as glutathione-S-transferase (GST) and glutathione reductase (GR). Molecular docking analysis suggests putative interactions of DISPOL with GST and GR which could account for its inhibitory effect on these enzymes. Further, DISPOL induced reductive environment preceded G1 arrest and apoptosis as evidenced by decreased expression of cell cycle genes (Cyclin D1 and Cyclin E1) and elevation of p21 and apoptotic markers (cleaved caspase 3 and cleaved PARP). The combinatorial experiments involving DISPOL and redox modulatory agents such as N-acetylcysteine (NAC) and buthionine sulfoximine (BSO) indeed confirmed the role of reductive stress in DISPOL-induced cell death. Finally, Lipinski's rule suggests attributes of drug likeness in DISPOL. Taken together, DISPOL exhibits a novel mechanism of reductive stress-mediated cell death in A549cells that warrants future exploration as anticancer agent.

Seleno-aspirin compound AS-10 promotes histone acetylation ahead of suppressing androgen receptor transcription, G1 arrest, and apoptosis of prostate cancer cells.

The novel selenium-aspirin compound AS-10 was recently reported by us with a cancer cell killing potency three orders of magnitude greater than aspirin in pancreatic cancer cell lines with caspase-mediated apoptosis and a reasonable selectivity against malignant cells. Although we also observed its cytocidal activity against PC-3 and DU145 androgen receptor (AR)-negative and P53-null/mutant aggressive human prostate cancer (PCa) cell lines in NCI-60 screen, the potential involvement and targeting of AR and P53 pathways that are intact in early-stage prostate carcinogenesis has not been examined, nor its primary molecular signaling after exposure. Human LNCaP PCa cells with functional AR and intact P53 were used to examine their cell cycle and cell fate responses to AS-10 exposure and upstream molecular signaling events including histone acetylation as a known aspirin effect. The AR-positive 22Rv1 human PCa cells were used to validate key findings. In addition to confirming AS-10's superior cytocidal potency than aspirin against all four PCa cell lines, we report a rapid (within 5 min) promotion of histone acetylation several hours ahead of the suppression of AR and prostate-specific antigen (PSA, coded by KLK3 gene) in LNCaP and 22Rv1 cells. AS-10 decreased AR and KLK3 mRNA levels without impacting pre-existing AR protein degradation or nuclear translocation in LNCaP cells. Sustained exposure to AS-10 arrested cells predominantly in G1 , and induced caspase-mediated apoptosis without necrosis. The death induced by AS-10 in LNCaP cells was attenuated by nontranscriptional activation of P53 protein or Jun N-terminal Kinase cellular stress signaling and was mitigated modestly by glutathione-boosting antioxidant N-acetylcysteine. AS-10 synergized with histone deacetylase inhibitor SAHA to suppress AR/PSA abundance and kill LNCaP cells. RNA-seq confirmed AR suppression at the transcriptional level and suggested multiple oncogene, cyclin, and CDK/CKI transcriptional actions to contribute to the cellular consequences. AS-10 promotes histone acetylation as its probable primary mechanism of action to induce PCa cell-cycle arrest and apoptosis, regardless of AR and P53 status. Nevertheless, the inhibition of AR signaling through mechanisms distinct from canonical AR antagonists may hold promise for combinatorial use with androgen deprivation therapy regimens or AR-axis targeting drugs.

Proliferation and Self-Renewal Are Differentially Sensitive to NRASG12V Oncogene Levels in an Acute Myeloid Leukemia Cell Line.

Different levels of activated NRAS may exert distinct effects on proliferation and self-renewal.

Anticancer Effects of ZnO/CNT@Fe3O4 in AML-Derived KG1 Cells: Shedding Light on Promising Potential of Metal Nanoparticles in Acute Leukemia.

Background: Therapeutic approaches for acute myeloid leukemia (AML) have remained largely unchanged for over 40 years and cytarabine and an anthracycline (e.g., daunorubicin) backbone is the main induction therapy for these patients. Resistance to chemotherapy is the major clinical challenge and contributes to short-term survival with a high rate of disease recurrence. Given the established efficacy of nanoparticles in cancer treatment, this study was designed to evaluate the anticancer property of our novel nanocomposite in the AML-derived KG1 cells. Materials and Methods: To assess the anti-leukemic effects of our nanocomposite on AML cells, we used MTT and trypan blue assays. Flow cytometric analysis and q-RT-PCR were also applied to evaluate the impact of nanocomposite on cell cycle and apoptosis. Results: Our results outlined that ZnO/CNT@Fe3O4 decreased viability and metabolic activity of KG1 cells through induction of G1 arrest by increasing the expression of p21 and p27 cyclin-dependent kinase inhibitors and decreasing c-Myc transcription. Moreover, ZnO/CNT@Fe3O4 markedly elevated the percentage of apoptotic cells which was coupled with a significant alteration of Bax and Bcl-2 expressions. Synergistic experiments showed that ZnO/CNT@Fe3O4 enhances the cytotoxic effects of Vincristine on KG1 cells. Conclusion: In conclusion, this study sheds light on the potent anti-leukemic effects of ZnO/CNT@Fe3O4 and provides evidence for the application of this agent in the treatment of acute myeloid leukemia.

LB1018 ALRN-6924, a dual inhibitor of MDMX and MDM2, protects human scalp hair follicles and their epithelial stem cells from paclitaxel-induced toxicity

Taxanes like paclitaxel (PTX) cause severe and often permanent chemotherapy-induced alopecia (CIA). We have previously shown that PTX damages human scalp hair follicles (HFs) by inducing massive mitotic defects and apoptosis in hair matrix (HM) keratinocytes as well as bulge stem cell DNA damage, and that pharmacological induction of transient G1 arrest can be HF- and stem cell-protective. We tested whether ALRN-6924 can protect human HFs from PTX-induced toxicity ex vivo. ALRN-6924 is a clinical-stage MDM2/MDMX dual inhibitor that activates p53 to upregulate p21, transiently arresting and selectively protecting healthy normal cells from chemotherapy in patients with p53-mutant cancers without protecting cancer cells.

C-myc-mediated upregulation of NAT10 facilitates tumor development via cell cycle regulation in non-small cell lung cancer.

N-acetyltransferase 10 (NAT10) is a nucleolar acetyltransferase and has been reported to facilitate tumorigenesis in various cancers, but its role in NSCLC and how it is regulated remain to be assessed. The expression of NAT10 was explored in online databases and our collected clinical specimens. The relationship of NAT10 and clinical characteristics was evaluated using the online databases. Functional analyses were utilized to determine the effect of NAT10 on the proliferation and migration abilities. KEGG pathway analyses were conducted to investigate NAT10-related pathways in NSCLC. The influence of NAT10 on cell cycle was assessed by flow cytometry and cell synchronization assay. The association between c-myc and NAT10 promoter was determined by ChIP. Compared with normal tissue, NAT10 was significantly overexpressed in NSCLC. Upregulated NAT10 was associated with more advanced stage for lung adenocarcinoma and shorter overall survival and first progression time for lung cancer. NAT10 could promote proliferation and migration of NSCLC cells in vitro. c-myc positively regulated the expression of NAT10 as a transcription factor. KEGG pathway analyses indicated that NAT10 was significantly involved in cell cycle regulation, cytokine-cytokine receptor interaction and other pathways. The knockdown of NAT10-induced G1 arrest, which was possibly mediated by the downregulation of cyclin D1.Our findings suggested that c-myc-mediated upregulation of NAT10 promoted the proliferation and migration of NSCLC cells and NAT10 might be a marker for prognosis and a promising target for treatment in NSCLC.

Low-Dose Rifabutin Increases Cytotoxicity in Antimitotic-Drug-Treated Resistant Cancer Cells by Exhibiting Strong P-gp-Inhibitory Activity

The cytotoxicity of various antibiotics at low doses in drug-resistant cancer cells was evaluated. Low doses of rifabutin were found to markedly increase the cytotoxicity of various antimitotic drugs, such as vincristine (VIC), to P-glycoprotein (P-gp)-overexpressing antimitotic-drug-resistant KBV20C cells. Rifabutin was also found to exert high levels of P-gp-inhibitory activity at 4 and 24 h posttreatment, suggesting that the cytotoxicity of VIC + rifabutin was mainly due to the direct binding of rifabutin to P-gp and the reduction of VIC efflux by P-gp. The combination of VIC + rifabutin also increased early apoptosis, G2 arrest, and the DNA damaging marker, pH2AX protein. Interestingly, only the combination of VIC + rifabutin induced remarkable levels of cytotoxicity in resistant KBV20C cells, whereas other combinations (VIC + rifampin, VIC + rifapentine, and VIC + rifaximin) induced less cytotoxicity. Such finding suggests that rifabutin specifically increases the cytotoxicity of VIC in KBV20C cells, independent of the toxic effect of the ansamycin antibiotic. Only rifabutin had high P-gp-inhibitory activity, which suggests that its high P-gp-inhibitory activity led to the increased cytotoxicity of VIC + rifabutin. As rifabutin has long been used in the clinic, repositioning this drug for P-gp-overexpressing resistant cancer could increase the availability of treatments for patients with drug-resistant cancer.

Mitochondrial phosphoenolpyruvate carboxykinase promotes tumor growth in estrogen receptor-positive breast cancer via regulation of the mTOR pathway.

Tumor cells may aberrantly express metabolic enzymes to adapt to their environment for survival and growth. Targeting cancer-specific metabolic enzymes is a potential therapeutic strategy. Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the conversion of oxaloacetate to phosphoenolpyruvate and links the tricarboxylic acid cycle and glycolysis/gluconeogenesis. Mitochondrial PEPCK (PEPCK-M), encoded by PCK2, is an isozyme of PEPCK and is distributed in mitochondria. Overexpression of PCK2 has been identified in many human cancers and demonstrated to be important for the survival program initiated upon metabolic stress in cancer cells. We evaluated the expression status of PEPCK-M and investigated the function of PEPCK-M in breast cancer. We checked the expression status of PEPCK-M in breast cancer samples by immunohistochemical staining. We knocked down or overexpressed PCK2 in breast cancer cell lines to investigate the function of PEPCK-M in breast cancer. PEPCK-M was highly expressed in estrogen receptor-positive (ER+ ) breast cancers. Decreased cell proliferation and G0 /G1 arrest were induced in ER+ breast cancer cell lines by knockdown of PCK2. PEPCK-M promoted the activation of mTORC1 downstream signaling molecules and the E2F1 pathways in ER+ breast cancer. In addition, glucose uptake, intracellular glutamine levels, and mTORC1 pathways activation by glucose and glutamine in ER+ breast cancer were attenuated by PCK2 knockdown. PEPCK-M promotes proliferation and cell cycle progression in ER+ breast cancer via upregulation of the mTORC1 and E2F1 pathways. PCK2 also regulates nutrient status-dependent mTORC1 pathway activation in ER+ breast cancer. Further studies are warranted to understand whether PEPCK-M is a potential therapeutic target for ER+ breast cancer.

NSUN2-mediated RNA m5C modification modulates uveal melanoma cell proliferation and migration

ABSTRACT RNA 5-methylcytosine (m5C) is a widespread post-transcriptional modification involved in diverse biological processes through controlling RNA metabolism. However, its roles in uveal melanoma (UM) remain unknown. Here, we describe the biological roles and regulatory mechanisms of RNA m5C in UM. Initially, we identified significantly elevated global RNA m5C levels in both UM cells and tissue specimens using ELISA assay and dot blot analysis. Meanwhile, NOP2/Sun RNA methyltransferase family member 2 (NSUN2) was upregulated in both types of these samples, whereas NSUN2 knockdown significantly decreased RNA m5C level. Such declines inhibited UM cell migration and suppressed cell proliferation through cell cycle G1 arrest. Furthermore, bioinformatic analyses, m5C-RIP-qPCR, and luciferase assay identified β-Catenin (CTNNB1) as a direct target of NSUN2-mediated m5C modification in UM cells. Additionally, overexpression of miR-124a in UM cells diminished NSUN2 expression levels indicating that it is an upstream regulator of this response. Our study suggests that NSUN2-mediated RNA m5C methylation provides a potential novel target to improve the therapeutic management of UM pathogenesis.

Structural Organization and Function of the Golgi Ribbon During Cell Division.

The Golgi complex has a central role in the secretory traffic. In vertebrate cells it is generally organized in polarized stacks of cisternae that are laterally connected by membranous tubules, forming a structure known as Golgi ribbon. The steady state ribbon arrangement results from a dynamic equilibrium between formation and cleavage of the membrane tubules connecting the stacks. This balance is of great physiological relevance as the unlinking of the ribbon during G2 is required for mitotic entry. A block of this process induces a potent G2 arrest of the cell cycle, indicating that a mitotic “Golgi checkpoint” controls the correct pre-mitotic segregation of the Golgi ribbon. Then, after mitosis onset, the Golgi stacks undergo an extensive disassembly, which is necessary for proper spindle formation. Notably, several Golgi-associated proteins acquire new roles in spindle formation and mitotic progression during mitosis. Here we summarize the current knowledge about the basic principle of the Golgi architecture and its functional relationship with cell division to highlight crucial aspects that need to be addressed to help us understand the physiological significance of the ribbon and the pathological implications of alterations of this organization.