Cell Cycle Disturbance Research Articles

Exposure to Benzo(a)pyrene promotes proliferation and inhibits differentiation of stromal cells in mice during decidualization

The environmental pollutant Benzo(a)pyrene (BaP) has an adverse effect on the reproductive performance of mammals. We previously showed that BaP treatment during early pregnancy damages endometrial morphology and impairs embryo implantation. Endometrial decidualization at the implantation site (IS) after embryo implantation is crucial for pregnancy maintenance and placental development. The balance between proliferation and differentiation in endometrial stromal cells (ESCs) is a crucial event of decidualization, which is regulated by the cell cycle. Here, we report that abnormal decidualization caused by BaP is associated with cell cycle disturbance of stromal cells. The mice in the treatment group were gavaged with 0.2 mg/kg/day BaP from day 1–8 of pregnancy, while those in control were gavaged with corn oil in parallel. BaP damaged the decidualization of ESCs and reduced the number of polyploid cells. Meanwhile, BaP up-regulated the expression of Ki67 and PCNA, affecting the differentiation of stromal cells. The cell cycle progression analysis during decidualization in vivo and in vitro showed that BaP induced polyploid cells deficiency with enhanced expressions of CyclinA(E)/CDK2, CyclinD/CDK4 and CyclinB/CDK1, which promote the transformation of cells from G1 to S phase and simultaneously activate the G2/M phase. The above results indicated that BaP exposure accelerates cell cycle progression, promotes ESC proliferation, inhibits differentiation, and impedes proper decidualization and polyploidy development. Thus, the imbalance of ESC proliferation and differentiation would be an important mechanism for BaP-induced defective decidualization.

PER2 integrates circadian disruption and pituitary tumorigenesis.

Rationale: The role of circadian clock in pituitary tumorigenesis remains elusive. Here we investigate whether and how circadian clock modulates the development of pituitary adenomas. Methods and Results: We found altered expression of pituitary clock genes in patients with pituitary adenomas. In particular, PER2 is prominently upregulated. Further, jetlagged mice with PER2 upregulation have accelerated growth of GH3 xenograft tumor. Conversely, loss of Per2 protects mice against developing estrogen-induced pituitary adenoma. Similar antitumor effect is observed for SR8278, a chemical that can decrease pituitary PER2 expression. RNA-seq analysis suggests involvement of cell cycle disturbance in PER2 regulation of pituitary adenoma. Subsequent in vivo and cell-based experiments validate that PER2 induces pituitary expression of Ccnb2, Cdc20 and Espl1 (three cell cycle genes) to facilitate cell cycle progression and inhibit apoptosis, thereby promoting pituitary tumorigenesis. Mechanistically, PER2 regulates the transcription of Ccnb2, Cdc20 and Espl1 through enhancing the transcriptional activity of HIF-1α. HIF-1α trans-activates Ccnb2, Cdc20 and Espl1 via direct binding to its specific response element in the gene promoters. Conclusion: PER2 integrates circadian disruption and pituitary tumorigenesis. These findings advance our understanding of crosstalk between circadian clock and pituitary adenomas and highlight the relevance of clock-based approaches in disease management.

Expression Profiles of CDKN2A, MDM2, E2F2 and LTF Genes in Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is one of the most commonly detected neoplasms worldwide. Not all mechanisms associated with cell cycle disturbances are known in OSCC. Examples of genes involved in the control of the cell cycle are CDKN2A, MDM2, E2F2 and LTF. The aim of this study was to examine the possible association between CDKN2A, MDM2, E2F2 and LTF mRNA expression and influence on clinical variables. The study group consisted of 88 Polish patients. The gene expression levels were assessed by quantitative reverse transcription PCR. We found no statistically significant differences in the expression level of CDKN2A, MDM2, E2F2 and LTF genes in tumour samples compared to margin samples. No association was found between the gene expression levels and clinical parameters, except E2F2. The patients with G2 tumours had a significantly higher gene expression level of E2F2 than patients with low-grade G1 tumours. We have not demonstrated that a change in expression profiles of genes has a significant impact on the pathogenesis of OSCC. It may also be useful to conduct further studies on the use of E2F2 expression profile changes as a factor to describe the invasiveness and dynamics of OSCC development.

Mitochondrial targeting half-sandwich iridium(III) and ruthenium(II) dppf complexes and in vitro anticancer assay

Considering the potential application of half-sandwich and ferrocenyl-containing organometallic complexes in the area of anticancer, four half-sandwich iridium(III) (IrIII) and ruthenium(II) (RuII) diphenylphosphino ferrocene (dppf) complexes were prepared in this study. Complexes showed favorable anti-proliferation activity towards A549 cell lines compared to cisplatin, meanwhile, which could effectively inhibit cell migration. These complexes followed an energy dependence uptake mechanism, effectively accumulated in mitochondria with a Pearson's Colocalization Coefficient (PCC) of 0.77, decreased the mitochondrial membrane potential, induced a surge of reactive oxygen species, disturbed cell cycle, and eventually led to apoptosis. Western blot assay further confirmed that these complexes induced apoptosis following a mitochondrial pathway. Above all, half-sandwich IrIII and RuII dppf complexes show the prospect of becoming a new multifunctional therapeutic platform for mitochondrial targeted imaging and anticancer drugs.

Dose-effect of polystyrene microplastics on digestive toxicity in chickens (Gallus gallus): Multi-omics reveals critical role of gut-liver axis

IntroductionMicroplastic pollution seriously threatens the health and safety of humans and wildlife. Avian is one of the main species endangered by microplastics. However, the damage mechanism of microplastics to the digestive system of avian is not clear. ObjectivesThe gut-liver axis is a bidirectional channel that regulates the exchange of information between the gut and the liver and is also a key target for tissue damage caused by pollutants. This study aimed to elucidate the digestive toxicity of microplastics in avian and the key role of the gut-liver axis in it. MethodsWe constructed an exposure model for microplastics in environmental concentrations and toxicological concentrations in chickens and reveal the digestive toxicity of polystyrene microplastics (PS-MPs) in avian by 16S rRNA, transcriptomics and metabolomics. ResultsPS-MPs changed the death mode from apoptosis to necrosis and pyroptosis by upregulating Caspase 8, disrupting the intestinal vascular barrier, disturbing the intestinal flora and promoting the accumulation of lipopolysaccharide. Harmful flora and metabolites were translocated to the liver through the liver-gut axis, eliciting hepatic immune responses and promoting hepatic lipid metabolism disorders and apoptosis. Liver injury involves multiple molecular effects of mitochondrial dynamics disturbance, oxidative stress, endoplasmic reticulum stress, and cell cycle disturbance. Furthermore, metabolomics suggested that caffeine and melanin metabolites may be potential natural resistance substances for microplastics. ConclusionTaken together, our data demonstrate the digestive damage of PS-MPs in avian, revealing a critical role of the liver-gut axis in it. This will provide a reference for protecting the safety of avian populations.

New gold(III) chlorophenyl terpyridine complex: Biomolecular interactions and anticancer activity against human oral squamous cell carcinoma

In this work we synthesized new monofunctional gold(III) complex [Au(Cl‐Ph‐tpy)Cl]Cl2 (Cl‐Ph‐tpy = 4′‐[4‐chlorophenyl]‐2,2′:6′, 2″‐terpyridine). This complex was characterized by UV–Vis, NMR, IR, and ESI‐MS spectrometry. The kinetic study of the substitution reactions of the Au‐Cl‐Ph‐tpy complex with biomolecules showed that the rate constants depend on the nature of the entering nucleophile. Based on the calculated values of entropy (∆H≠ > 0) and enthalpy (∆S≠ < 0) the proposed substitution mechanism is associative. Additionally, the relative stability and thermodynamic properties of Au‐Cl‐Ph‐tpy complex were compared with the analogue Au‐tpy complex by the B3LYP/def2‐svp method. DNA/BSA binding studies showed that Au‐Cl‐Ph‐tpy complex interacts with CT DNA through the intercalation and moderately quenches the fluorescence of tryptophan residues in serum albumin (BSA). Molecular docking confirmed results obtained by spectroscopic experiments and suggested site I (subdomain IIA) for binding of Au complex to BSA. We demonstrated that the Au chlorophenyl terpyridine complex possessed significant in vitro cytotoxic activity against human oral squamous carcinoma cells (CAL‐27), induced apoptosis, inhibited proliferation of CAL‐27 cells, and induced cell cycle disturbance. Treatment of CAL‐27 cells with the Au complex enhanced expression of cyclin‐dependent kinase inhibitors p21 and p27, resulting in cell cycle arrest in the G1 phase, reduced the percentage of CAL‐27 cells in S phase and decreased expression of Ki‐67. Additionally, Au complex reduced expression of phosphorylated STAT3 and downstream regulated molecules associated with cancer stemness, NANOG, and Sox2 protein.

In vitro anti-melanoma effect of polyphenolic compounds

Objective: To evaluate the effects of phenolic acids (caffeic, ferulic, and coumaric acids) and flavones (luteolin and apigenin) on the proliferation and melanogenesis in murine melanoma B16-F10 cells. Methods: Cell proliferation was determined after 24 and 48 hours of incubation using MTT assay. The effects of these tested compounds on cell cycle progression were analyzed by flow cytometry. Moreover, the melanin content and tyrosinase activity were measured spectrophotometrically at 475 nm. Results: Luteolin and apigenin exhibited significant anti-proliferative activity against B16-F10 cells, while caffeic, ferulic, and coumaric acids induced slight inhibition after 24 and 48 hours of incubation. The tested compounds disturbed cell cycle progression of B16-F10, by a subsequent decrease in G1 and arrested cycle progression in either G1/S or G2/M phase. Furthermore, apigenin provoked an increase in melanin content of B16-F10 cells. In contrast, luteolin, caffeic, ferulic and coumaric acids induced a decrease in melanin content of B16-F10 cells by inhibiting tyrosinase activity. Conclusions: These active polyphenols may be used as skin whitening agents or natural tanning agents to treat skin pigmentation disorders.

Overexpression of CYP11A1 recovers cell cycle distribution in renal cell carcinoma Caki-1

BackgroundClear cell renal carcinoma is commonly known for its metastasis propensity to outspread to other organs and is asymptomatic in the early stage. Recent studies have shown that deficiencies in CYP11A1 expression can lead to fatal adrenal failure if left untreated and are associated with downstream regulation in various cancer types. However, the molecular mechanisms of CYP11A1 and kidney cancer proliferation remain unclear.MethodsNormal and renal carcinoma cell lines (HEK293 and Caki-1) were transfected with plasmid encoding CYP11A1 to overexpress the P450scc protein. Cell cycle distribution was investigated using flow cytometry. The expression of proteins related to C-Raf/ERK/JNK/p38 signaling pathways was examined using western blot.ResultsWe observed that CYP11A1 overexpression suppressed the cyclin B1 and cell-division cycle 2 expression while cyclin-dependent kinases 2 and 4 were unaffected. Cancer cell migration and invasion were suppressed along with epithelial-intermediate metastatic markers Snail and Vimentin. In addition, in CYP11A1-overexpressing Caki-1 cells, cdc2/cyclinB1 was downregulated while the phosphorylation of cdc25c, a G2/M arrest-related upstream signal, was increased. The intrinsic-mitochondrial apoptosis markers were not significantly altered. We also identified that the C-Raf/ERK/JNK/p38 pathway is an important pro-apoptotic mechanism in CYP11A1-overexpressing cell-based models. Our results suggest that CYP11A1 overexpression recovered the disturbed cell cycle arrest distribution in renal carcinoma cell line Caki-1 through G2/M arrest and C-Raf/ERK/JNK pathway.ConclusionsOur findings may suggest promising new therapeutic targets to suppress kidney cancer proliferation without affecting normal cells, eventually improving the survival of patients with cancer.

Cell cycle disturbances in mucopolysaccharidoses: Transcriptomic and experimental studies on cellular models.

Mucopolysaccharidoses (MPS) are a group of lysosomal storage diseases caused by defects in genes coding for proteins involved in degradation of glycosaminoglycans (GAGs). These complex carbohydrates accumulate in cells causing their serious dysfunctions. Apart from the physical GAG storage, secondary and tertiary changes may contribute significantly to the pathomechanism of the disease. Among processes which were not systematically investigated in MPS cells to date there is the cell cycle. Here, we studied perturbances in this crucial cellular process in majority of MPS types. Transcriptomic analyses indicated that expression of many genes coding for proteins involved in the cell cycle is dysregulated in all tested MPS cells. Importantly, levels of transcripts of particular genes were changed in the same manner (i.e. either up- or down-regulated) in most or all types of the disease, indicating a common mechanism of the dysregulation. Flow cytometric studies demonstrated that the cell cycle is disturbed in all MPS types, with increased fractions of cells in the G0/G1 phase in most types and decreased fractions of cells in the G2/M phase in all types. We found that increased levels of cyclin D1 and disturbed timing of its appearance during the cell cycle may contribute to the mechanism of dysregulation of this process in MPS. Reduction of GAG levels by either a specific enzyme or genistein-mediated inhibition of synthesis of these compounds improved, but not fully corrected, the cell cycle in MPS fibroblasts. Therefore, it is suggested that combination of the therapeutic approaches devoted to reduction of GAG levels with cyclin D1 inhibitors might be considered in further works on developing effective treatment procedures for MPS.

The Pt(S-pr-thiosal)2 and BCL1 Leukemia Lymphoma: Antitumor Activity In Vitro and In Vivo.

B cell malignancies are, despite the development of targeted therapy in a certain percentage of the patients still a chronic disease with relapses, requiring multiple lines of therapy. Regimens that include platinum-based drugs provide high response rates in different B cell lymphomas, high-risk chronic lymphocytic leukemia (CLL), and devastating complication of CLL, Richter’s syndrome. The aim of this study was to explore the potential antitumor activity of previously synthetized platinum(IV) complex with alkyl derivatives of thyosalicilc acid, PtCl2(S-pr-thiosal)2, toward murine BCL1 cells and to delineate possible mechanisms of action. The PtCl2(S-pr-thiosal)2 reduced the viability of BCL1 cells in vitro but also reduced the growth of metastases in the leukemia lymphoma model in BALB/c mice. PtCl2(S-pr-thiosal)2 induced apoptosis, inhibited proliferation of BCL1 cells, and induced cell cycle disturbance. Treatment of BCL1 cells with PtCl2(S-pr-thiosal)2 inhibited expression of cyclin D3 and cyclin E and enhanced expression of cyclin-dependent kinase inhibitors p16, p21, and p27 resulting in cell cycle arrest in the G1 phase, reduced the percentage of BCL1 cells in the S phase, and decreased expression of Ki-67. PtCl2(S-pr-thiosal)2 treatment reduced expression of phosphorylated STAT3 and downstream-regulated molecules associated with cancer stemness and proliferation, NANOG, cyclin D3, and c-Myc, and expression of phosphorylated NFκB in vitro and in vivo. In conclusion, PtCl2(S-pr-thiosal)2 reduces STAT3 and NFκB phosphorylation resulting in inhibition of BCL1 cell proliferation and the triggering of apoptotic cell death.

Decreased junctional adhesion molecule 3 expression induces reactive oxygen species production and apoptosis in trophoblasts†.

Junctional adhesion molecule 3 (JAM3) is involved in epithelial cell junction, cell polarity, and motility. The molecular mechanisms underlying the role of JAM3 in placental dysfunction remain unclear. We hypothesized that JAM3 expression regulates trophoblast fusion, differentiation, proliferation, and apoptosis. Our results revealed that JAM3 was expressed in the cytotrophoblasts and syncytiotrophoblasts of first-trimester and term placental villi. JAM3 expression in cell-cell junctions decreased with the formation of syncytiotrophoblasts. Using trophoblasts as an in vitro model, we observed that forskolin and JAM3 knockdown significantly reduced JAM3 expression and increased syncytium formation. JAM3 knockdown additionally inhibited trophoblast proliferation and increased the number of trophoblasts in the sub-G1 and G2/M phases, indicating cell-cycle disturbance and apoptosis. Cell-cycle arrest was associated with the engagement of checkpoint kinase 2-cell division cycle 25C-cyclin-dependent kinase 1/cyclin B1 signaling. Increased expression of BIM, NOXA, XAF1, cytochrome c, and cleaved caspase-3 further indicated trophoblast apoptosis. Overexpression of JAM3 or recombinant JAM3 protein enhanced trophoblast adhesion and migration, which were inhibited by JAM3 knockdown. JAM3 knockdown induced reactive oxygen species and syncytin 2 expression in trophoblasts. Furthermore, H2O2-induced oxidative stress reduced JAM3 expression in trophoblasts and cell culture supernatants. H2O2 simultaneously induced trophoblast apoptosis. JAM3 expression was significantly decreased in the plasmas and placentas of patients with early-onset severe preeclampsia. Thus, our results show that JAM3 may not only be a structural component of trophoblast cell junctions but also regulates trophoblast fusion, differentiation, proliferation, apoptosis, and motility. Dysregulated trophoblast JAM3 expression is crucial in preeclampsia development.

Acetaminophen-induced reduction of NIMA-related kinase 7 expression exacerbates acute liver injury

Background & AimsAcetaminophen (APAP)-induced acute liver injury (ALI) is a global health issue characterised by an incomplete understanding of its pathogenesis and unsatisfactory therapies. NEK7 plays critical roles in both cell cycle regulation and inflammation. In the present study, we investigated the role and mechanism of NEK7 in APAP-induced ALI.MethodsIn mice with NEK7 overexpression (hydrodynamic tail vein injection of NEK7 plasmids), hepatocyte-specific NEK7 knockout (cKO), and inducible NEK7 knockout (iKO), an overdose of APAP was administered to induce ALI. Liver injury was determined by an analysis of serum liver enzymes, pathological changes, inflammatory cytokines, and metabonomic profiles. In vitro, hepatocyte damage was evaluated by an analysis of cell viability, the reactive oxygen species levels, and mitochondrial function in different cell lines. Hepatocyte proliferation and the cell cycle status were determined by Ki-67 staining, EdU staining, and the cyclin levels.ResultsNEK7 was markedly downregulated in APAP-induced injured liver and damaged hepatocytes. NEK7 overexpression in the liver significantly alleviated APAP-induced liver injury, as shown by the restored liver function, reduced pathological injury, and decreased inflammation and oxidative stress, which was confirmed in a hepatocyte cell line. Moreover, both NEK7 cKO and iKO mice exhibited exacerbation of APAP-induced ALI. Finally, we determined that cyclin B1-mediated cell cycle progression could mediate the protective effect of NEK7 against APAP-induced ALI.ConclusionsReduced NEK7 contributes to APAP-induced ALI, possibly by dysregulating cyclins and disturbing cell cycle progression.Lay summaryAcetaminophen-induced acute liver injury is one of the major global health issues, owing to its high incidence, potential severity, and limited therapeutic options. Our current understanding of its pathogenesis is incomplete. Herein, we have shown that reduced NEK7 (a protein with a key role in the cell cycle) exacerbates acetaminophen-induced acute liver injury. Hence, NEK7 could be a possible therapeutic target for the prevention or treatment of this condition.

Synthesis and Biological Activity Screening of Newly Synthesized Trimethoxyphenyl-Based Analogues as Potential Anticancer Agents.

A group of novel trimethoxyphenyl (TMP)-based analogues were synthesized by varying the azalactone ring of 2-(3,4-dimethoxyphenyl)-4-(3,4,5-trimethoxybenzylidene)oxazolone 1 and characterized using NMR spectral data as well as elemental microanalyses. All synthesized compounds were screened for their cytotoxic activity utilizing the hepatocellular carcinoma (HepG2) cell line. Compounds 9, 10 and 11 exhibited good cytotoxic potency with IC50 values ranging from 1.38 to 3.21 μM compared to podophyllotoxin (podo) as a reference compound. In addition, compounds 9, 10 and 11 exhibited potent inhibition of β-tubulin polymerization. DNA flow cytometry analysis of compound 9 shows cell cycle disturbance at the G2/M phase and a significant increase in Annexin-V-positive cells compared with the untreated control. Compound 9 was further studied regarding its apoptotic potential in HepG2 cells; it decreased the level of MMP and Bcl-2 as well as boosted the level of p53 and Bax compared with the control HepG2 cells.

Kinesin KIF15 regulates tubulin acetylation and spindle assembly checkpoint in mouse oocyte meiosis.

Microtubule dynamics ensure multiple cellular events during oocyte meiosis, which is critical for the fertilization and early embryo development. KIF15 (also termed Hklp2) is a member of kinesin-12 family motor proteins, which participates in Eg5-related bipolar spindle formation in mitosis. In present study, we explored the roles of KIF15 in mouse oocyte meiosis. KIF15 expressed during oocyte maturation and localized with microtubules. Depletion or inhibition of KIF15 disturbed meiotic cell cycle progression, and the oocytes which extruded the first polar body showed a high aneuploidy rate. Further analysis showed that disruption of KIF15 did not affect spindle morphology but resulted in chromosome misalignment. This might be due to the reduced stability of the K-fibers, which further induced the loss of kinetochore-microtubule attachment and activated spindle assembly checkpoint, showing with the failed release of Bub3 and BubR1. Based on mass spectroscopy analysis and coimmunoprecipitation data we showed that KIF15 was responsible for recruiting HDAC6, NAT10 and SIRT2 to maintain the acetylated tubulin level, which further affected tubulin acetylation for microtubule stability. Taken together, these results suggested that KIF15 was essential for the microtubule acetylation and cell cycle control during mouse oocyte meiosis.

Nivalenol affects Cyclin B1 level and activates SAC for cell cycle progression in mouse oocyte meiosis.

ObjectivesNivalenol (NIV) is a secondary metabolite of type B trichothecene mycotoxin produced by Fusarium genera, which is widely found in contaminated food and crops such as corn, wheat and peanuts. NIV is reported to have hepatotoxicity, immunotoxicity, genotoxicity, and reproductive toxicity. Previous studies indicate that NIV disturbs mammalian oocyte maturation. Here, we reported that delayed cell cycle progression might be the reason for oocyte maturation defect caused by NIV exposure.Methods and ResultsWe set up a NIV exposure model and showed that NIV did not affect G2/M transition for meiosis resumption, but disrupted the polar body extrusion of oocytes. Further analysis revealed that oocytes were arrested at metaphase I, which might be due to the lower expression of Cyclin B1 after NIV exposure. After cold treatment, the microtubules were disassembled in the NIV‐exposed oocytes, indicating that NIV disrupted microtubule stability. Moreover, NIV affected the attachment between kinetochore and microtubules, which further induced the activation of MAD2/BUBR1 at the kinetochores, suggesting that spindle assemble checkpoint (SAC) was continuously activated during oocyte meiotic maturation.ConclusionsTaken together, our study demonstrated that exposure to NIV affected Cyclin B1 expression and activated microtubule stability‐dependent SAC to ultimately disturb cell cycle progression in mouse oocyte meiosis.

Abstract 1694: Cytotoxic conditions alter cell free DNA concentration and fragment size in cancer cells

Abstract Introduction: Detection of circulating free DNA (cfDNA) is a promising tool to monitor and predict tumor progression and treatment efficacy. Our group has shown the clinical relevance of cfDNA as a biomarker of tumor development and progression in several cancer models, including colorectal carcinoma and melanoma. cfDNA may stem from a combination of apoptosis, necrosis, and cellular secretions. However, the source and mechanism of cfDNA release in cancer cells are still unclear. Indeed, while some data state that cfDNA is derived from apoptosis, other studies report it is mainly a product of cellular secretions. To better understand cfDNA release and its correlation with tumor biology, this study aimed to assess cfDNA kinetics under cell death, cell cycle arrest, and senescence by using an in vitro cancer model. Methods: Colorectal (HTC116 and HT29), lung (A549), and melanoma (MP41 and OMM2.5) cancer cells underwent cytotoxic conditions by drugs: TRIAL/APO2L, Roscovitine, and valproic acid (VAP), as well as environmental stress by heat and irradiation. Cell death and cell cycle were evaluated by FACS. β-galactosidase staining assessed senescence. cfDNA was extracted from 3 mL supernatant using QIAamp nucleic acid kit and evaluated by digital droplet PCR through hot spot mutations (Mut), and a mitochondrial gene (mt-cfDNA). Isolated cfDNA was visualized by fragment size using Bioanalyzer 2100. Results: Mut and mt-cfDNA increased during cytotoxic conditions in a dose-dependent manner. Cells under apoptosis released greater mut and mt-cfDNA compared to necrotic and untreated cells. Similarly, cell cycle disturbances by VAP were associated with an increase in Mut and mt-cfDNA. Moreover, β-galactosidase positive cells (5D post-irradiation) showed lower Mut and mt-cfDNA levels than control conditions. Electropherogram images showed that cytotoxic conditions alter fragment size distribution. While fragments about 100-200 bp were observed in apoptosis, necrosis showed more abundant fragments about >1000 bp. Conclusions: The release of cfDNA is influenced by cytotoxic and stress conditions. Knowing the biology of cfDNA can help understand its role in cancer development and how best to utilize this marker clinically, especially post cytotoxic agents that induce different cell death mechanisms. These findings have major implications in the interpretation of ctDNA in liquid biopsy as a biomarker of treatment response and tumor progression. Citation Format: Prisca Bustamante, Thupten Tsering, Julia Valdemarin Burnier. Cytotoxic conditions alter cell free DNA concentration and fragment size in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1694.

Design, Synthesis, In Vitro Biological Activity Evaluation and Stabilized Nanostructured Lipid Carrier Formulation of Newly Synthesized Schiff Bases-Based TMP Moieties.

A series of novel Schiff bases-based TMP moieties have been designed and synthesized as potential anticancer agents. The target Schiff bases were screened for their cytotoxic activity against the MDA-MB-231 breast cancer cell line. Most of the tested molecules revealed good cytotoxic activity, especially compounds 4h, 4j and 5d. Being the most potent, compound 4h showed good tubulin polymerization inhibition activity as revealed by immunofluorescence analysis and ELISA assay. Additionally, compound 4h was screened for cell cycle disturbance and apoptosis induction. Pre-G1 apoptosis and cell growth halt at the G2/M phase were discovered to be caused by it. Moreover, compound 4h induced apoptosis via p53 and Bax activation, as well as reduced the level of Bcl-2. Additionally, the most potent compound 4h was lodged on nanostructured lipid carriers (NLCs). 23 full factorial design was involved to govern the influence of the fabrication variables on the in vitro characters of the casted NLCs. F3 was picked as the optimum formula exhibiting dominant desirability value 0.805, EE% 95.6 ± 2.4, PS 222.4 ±18.7, PDI 0.23 ± 0.05 and ZP −39.2 ± 3.9 Mv. Furthermore, F3 affirmed improved solubility and release over the drug suspension. In the comparative cytotoxic activity, F3 was capable of diminishing the IC50 by around 2.15 times for pure 4h, while nearly close to the IC50 of the reference drug. Thus, NLCs could be a potential platform for boosted antitumor activity.

Pracaxi oil affects xenobiotic metabolisms, cellular proliferation, and oxidative stress without cytotogenotoxic effects in HepG2/C3A cells

Pentaclethra macroloba (Willd.) Kuntze seeds oil has been used as a topical healing agent, applied mainly to parturients and snake bites. The objective was to investigate the effects of pracaxi oil (POP) on HepG2/C3A cells under cytogenotoxicity, cell cycle and apoptosis influence, and expression of metabolism and other related cell types proliferation genes. Cytotoxicity was analyzed by MTT test and apoptosis and cell cycle interferences by flow cytometry. To identify genotoxicity were used comet and micronucleus tests. RT-qPCR investigated gene expression. PO chemical characterization has shown two significant triterpenes, identified as oleanolic acid and hederagenin. The results showed that the PO did not reduce cell viability at concentrations ranging from 31 to 500 μg/ml. Comet and micronucleus assays revealed the absence of genotoxic effects, and flow cytometry showed no cell cycle or apoptosis disturbance. RT-qPCR indicated that PO up-regulated genes related to metabolism (CYP3A4, CYP1A2, CYP1A1), cell proliferation (mTOR), and oxidative stress (GPX1). The data indicate that PO has no cytogenotoxic effects and suggest that it activated the PI3/AKT/mTOR cascade of cell growth and proliferation. Inside the cells, the PO activated xenobiotic metabolizing genes, responsible for reactive oxygen species (ROS) generation, can neutralize ROS with increased GPX1 gene expression without genetic damage, interruption of the cell cycle, or induction of apoptosis.

Lysosomal Targeted Cyclometallic Iridium(Ⅲ) Salicylaldehyde-Coumarin Schiff Base Complexes and Anticancer Application.

Natural coumarin derivatives and cyclometallic iridium (Ⅲ) (IrⅢ) complexes have attracted much attention in the field of anticancer. In this study, six coumarin-modified cyclometallic IrⅢ salicylaldehyde Schiff base complexes ([(ppy)2Ir(O^N)]/[(ppy-CHO)2Ir(O^N)]) were designed and synthesized. Compared with coumarin and IrⅢ complex monomers, target complexes exhibited favorable cytotoxic activity toward A549 and BEAS-2B cells. These complexes could induce extensive apoptosis of A549 cell (late apoptosis), which was represented by the disturbance of cell cycle (G1-phase) and the accumulation of intracellular reactive oxygen species, exhibiting an anticancer mechanism of oxidation. With the help of suitable fluorescence of these complexes, no conflict with the probes, confocal detection confirmed that complexes showed an energy-dependent cellular uptake mechanism and triggered lysosome-mediated apoptosis in A549 cell line. Above all, our findings reveal the design of a lysosomal targeting cyclometallic IrⅢ Schiff base complexes and provide a new idea for the design of integrated drugs for diagnosis and treatment.

Highly Cytotoxic Osmium(II) Compounds and Their Ruthenium(II) Analogues Targeting Ovarian Carcinoma Cell Lines and Evading Cisplatin Resistance Mechanisms.

(1) Background: Ruthenium and osmium complexes attract increasing interest as next generation anticancer drugs. Focusing on structure-activity-relationships of this class of compounds, we report on 17 different ruthenium(II) complexes and four promising osmium(II) analogues with cinnamic acid derivatives as O,S bidentate ligands. The aim of this study was to determine the anticancer activity and the ability to evade platin resistance mechanisms for these compounds. (2) Methods: Structural characterizations and stability determinations have been carried out with standard techniques, including NMR spectroscopy and X-ray crystallography. All complexes and single ligands have been tested for cytotoxic activity on two ovarian cancer cell lines (A2780, SKOV3) and their cisplatin-resistant isogenic cell cultures, a lung carcinoma cell line (A549) as well as selected compounds on three non-cancerous cell cultures in vitro. FACS analyses and histone γH2AX staining were carried out for cell cycle distribution and cell death or DNA damage analyses, respectively. (3) Results: IC50 values show promising results, specifically a high cancer selective cytotoxicity and evasion of resistance mechanisms for Ru(II) and Os(II) compounds. Histone γH2AX foci and FACS experiments validated the high cytotoxicity but revealed diminished DNA damage-inducing activity and an absence of cell cycle disturbance thus pointing to another mode of action. (4) Conclusion: Ru(II) and Os(II) compounds with O,S-bidentate ligands show high cytotoxicity without strong effects on DNA damage and cell cycle, and this seems to be the basis to circumvent resistance mechanisms and for the high cancer cell specificity.