Chondroitin sulfate-functionalized selenium nanoparticle-induced S-phase cell cycle arrest and apoptosis in HeLa Cells.

This study aimed to evaluate the anti-cervical cancer activity of chondroitin sulfate-functionalized selenium nanoparticles (SeCS) and to elucidate their action mechanism. Cytotoxic effect of SeCS on HeLa cells was assessed by MTT assay. Further molecular mechanism of SeCS was analyzed by flow cytometric assay and western blotting. The results showed that treatment with SeCS resulted in a dose- and time-dependent inhibition in the proliferation of HeLa cells. The data obtained from flow cytometry demonstrated that SeCS inhibited HeLa cell growth via the induction of S-phase arrest and cell apoptosis. Further mechanism analysis found that SeCS down-regulated expression levels of cyclin A and CDK2 and up-regulated p21 expression, which contributed to S arrest. Moreover, SeCS increased the level of Bax and decreased the expression of Bcl-2, resulting in the release of cytochrome C from mitochondria and activating caspase-3/8/9 for caspase-dependent apoptosis. Meanwhile, intracellular reactive oxygen species (ROS) levels were elevated after SeCS treatment, suggesting that ROS might be upstream of SeCS-induced S-phase arrest and cell apoptosis. These data show that SeCS has anti-tumor effects and possesses the potential to become a new therapeutic agent or adjuvant therapy for cancer patients. PRACTICAL APPLICATION: In our previous study, we used chondroitin sulfate to stabilize nano-selenium to obtain SeCS to improve the bioactivity and stability of nano-selenium. We found that it possessed an inhibitory effect on HeLa cells. However, the molecular mechanism remains unclear. This study elucidated the mechanism of SeCS damage to HeLa cells. SeCS has the potential to become a new therapeutic agent or adjuvant therapy for cancer patients.

Abnormal expression of H-Ras induces S-phase arrest and mitotic catastrophe in human T-lymphocyte leukemia.

Background: Leukemia is a neoplasm with high incidence and mortality rates. Mitotic death has been observed in tumor cells treated with chemotherapeutic agents. Ras family proteins participate in the transduction of signals involved in different processes, such as proliferation, differentiation, survival, and paradoxically, initiation of cell death. Methods: This study investigated the effect of H-Ras expression on human T-cell acute lymphoblastic leukemia MOLT-4 cells. Cells were electroporated with either wild-type (Raswt) or oncogenic mutant in codon 12 exon 1 (Rasmut) versions of H-Ras gene and stained for morphological analysis. Cell viability was assessed using trypan blue staining and cell cycle analysis using flow cytometry. H-Ras gene expression was determined using quantitative real-time reverse transcription polymerase chain reaction. The t, ANOVA, and Scheffe tests were used for statistical analysis. Results: Human T-cell acute lymphoblastic leukemia MOLT-4 cells showed nuclear fragmentation and presence of multiple nuclei and micronuclei after transfection with either wt or mutant H-Ras genes. Cell cycle analysis revealed a statistically significant increase in cells in the S phase when transfected with either wt (83.67%, P<0.0005) or mutated (81.79%, P<0.0001) H-Ras genes. Although similar effects for both versions of H-Ras were found, cells transfected with the mutated version died at 120 h of mitotic catastrophe. Conclusion: Transfection of human T-cell acute lymphoblastic leukemia MOLT-4 cells with either normal or mutated H-Ras genes induced alterations in morphology, arrest in the S phase, and death by mitotic catastrophe.

Alcohol extracts of Chinese bayberry branch induce S-phase arrest and apoptosis in HepG2 cells.

The alcohol extracts of Chinese bayberry (Myrica rubra) branches (MRBE) are rich in flavonoids which have a variety of medicinal benefits, but their effects on human HepG2 were unknown. In this study, the effects of MRBE on HepG2 cell growth and its potential for inhibiting cancer were explored. The results displayed that MRBE inhibited HepG2 proliferation both by arresting cells in S phase and promoting apoptosis. Quantitative reverse-transcription PCR (qRT-PCR), western blotting, and immunofluorescence showed that MRBE induced S-phase arrest by upregulating p21, which in turn downregulated cyclin and cyclin-dependent kinase messenger RNA (mRNA) and protein. Apoptosis was induced by blocking the expression of BCL-2 and suppression of the Raf/ERK1 signaling pathways. These results indicated that MRBE may have the potential for treatment of human liver cancer, highlighting novel approaches for developing new pharmacological tools for the treatment of this deadly type cancer. Meanwhile, it provides a new direction for the medicinal added values of Chinese bayberry, which helped to broaden the diversified development of its industry chain.

Ghrelin enhances cisplatin sensitivity in HO-8910 PM human ovarian cancer cells.

BACKGROUND: Resistance to platinum-based chemotherapy is one of the crucial problems in ovarian cancer treatment. Ghrelin, a widely distributed peptide hormone, participates in a series of cancer progression. The aim of this study is to determine whether ghrelin influences the sensitivity of ovarian cancer to cisplatin, and to demonstrate the underlying mechanism. METHODS: The anti-tumor effects of ghrelin and cisplatin were evaluated with human ovarian cancer cells HO-8910 PM in vitro or in vivo. Cell apoptosis and cell cycle were analyzed via flow cytometry assay. The signaling pathway and the expression of cell cycle protein were analyzed with Western Blot. RESULTS: Our results showed that treatment with ghrelin specifically inhibited cell proliferation of HO-8910 PM and sensitized these cells to cisplatin via S phase cell cycle arrest, and enhanced the inhibitory effect of cisplatin on tumor growth of HO-8910 PM derived xenografts in vivo. Treatment with ghrelin inhibited the expression of p-Erk1/2 and p-p38, which was opposite the effect of cisplatin. However, under the treatment of ghrelin, cisplatin treatment exhibited a stronger effect on inhibiting P21 expression, upregulating p-CDK1 and cyclin B1 expression, and blocking cell cycle progression. Mechanistically, ghrelin promoted S phase cell cycle arrest and upregulated p-CDK1 and cyclin B1 expression induced by cisplatin via inhibition of p38. CONCLUSION: This study revealed a specifically inhibitory effect of ghrelin on platinum-resistance via suppressing p-P38 and subsequently promoting p-CDK1 mediated cell cycle arrest in HO-8910 PM.

Extracellular ATP Induced S-Phase Cell Cycle Arrest via P2Y Receptor-Activated ERK Signaling in Poorly Differentiated Oral Squamous Cell Carcinoma SAS Cells.

Extracellular ATP in the tumor microenvironment exhibits either pro- or antitumor effect via interaction with P2Y receptors, but the intracellular signaling and functional roles of P2Y receptors in oral squamous cell carcinoma (OSCC) are unclear. We aimed to study the effect of ATP on OSCC cell lines and the potential mechanisms involved. Through GEPIA dataset analysis, high expression levels of mRNA encoding P2Y receptors, the ATP-induced G protein-coupled receptors, were associated with better overall patient survival in head and neck squamous cell carcinoma. qPCR analysis showed that the poorly differentiated OSCC SAS cell line, had higher P2RY1 expression level compared to the well-differentiated H103 and H376 cell lines. Western blotting and flow cytometry analyses revealed that ATP phosphorylated ERK and elevated intracellular calcium signaling in all tested cell lines. A significant S-phase cell cycle arrest was observed in SAS, and preincubation with the MEK inhibitor PD0325901 reversed the ATP-induced S-phase arrest. We further demonstrated that ATP induced a slight reduction in cell count and colony formation yet significant apoptosis in SAS. Overall, we postulate that the ATP-induced S-phase arrest effect in SAS cells may be regulated through P2Y receptor-mediated ERK signaling, thus suggesting a potential antitumor effect of ATP via interaction with its distinct profile of P2Y receptors.

MAGT1 is required for HeLa cell proliferation through regulating p21 expression, S-phase progress, and ERK/p38 MAPK MYC axis.

Magnesium transporter subtype 1 (MAGT1) is known to participate in animal development and cell differentiation. Thus far, MAGT1 studies have mainly focused on its role in cardiomyocyte regulation and differentiation; only a few studies have demonstrated its role in cell proliferation. To investigate the underlying mechanism of MAGT1 in cell proliferation, HeLa and SiHa cells were transiently knocked down with different siRNAs. We showed that cell proliferation was substantially restricted by S-phase arrest and apoptosis in the MAGT1-knocked down cells, which was further confirmed by the increased expression of p21, cyclin-A1, and cyclin-B1, as well as the decreased expression of MYC, cyclin-D1, cyclin-E1, and CDK2. MAGT1 knockdown also resulted in significant changes in the transcriptional expression of 1,598 genes that were analyzed by RNA sequencing. Bioinformatics analysis showed that MAGT1 was related to the MAPK signaling pathway. Western blot analysis confirmed that the phosphorylation of extracellular signal-related protein kinase 1/2 (ERK1/2) and p38 was remarkably reduced in MAGT1 down-regulated groups. Additionally, MAGT1 was required for the function of viral proteins E6/E7 during cell proliferation and G1/S cell-cycle progression. Therefore, MAGT1 plays a crucial role in the proliferation of HPV-positive cervical cancer cells, S-phase progression, and the ERK/p38 MAPK signaling pathway. These results indicate the potential of MAGT1 as a novel target for anticancer research.Abbreviations: MAGT1: Magnesium transporter subtype 1; MAPK: Mitogen-activated protein kinase; XMEN: X-linked immunodeficiency with Magnesium defect, Epstein-Barr virus infection and Neoplasia; BMMSCs: Bone Marrow Mesenchymal Stem Cells; Dpp: Decapentaplegic; CDKIs: CDK inhibitors; GPCR: G-protein coupled receptor; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; RTK: Receptor Tyrosine Kinase; PTK: Protein Tyrosine Kinase; FGFR: Fibroblast Growth Factor Receptor; BMP: Bone Morphogenetic Protein; HPV18 E6/E7: Human Papillomavirus 18 Early protein 6/ early protein 7; FACS: Fluorescence Activated Cell Sorting; PI: Propidium Iodide.

Synergistic inhibitory effects of the oxyresveratrol and dacarbazine combination against melanoma cells.

Various therapies have been developed to target malignant melanoma, which is associated with a high mortality rate worldwide. Although dacarbazine (DTIC) is employed for treating melanoma, it is associated with several side effects. Hence, patients with melanoma are co-treated with additional drugs to mitigate the side effects of DTIC. In the present study, synergistic therapeutic effects of the DTIC/oxyresveratrol (ORT) combination were examined using the human malignant melanoma WM-266-4 cell line. Treatment with ORT and DTIC inhibited the proliferation of WM-266-4 cells. Compared with those in the ORT- and DTIC-treated groups, the proportion of cells arrested at the S phase, as well as apoptotic rates, were increased in the ORT and DTIC co-treatment group. In WM-266-4 cells, synergistic proliferation-inhibitory activities of the ORT/DTIC combination were assessed based on cell viability and migration, antioxidant capacity, cytokine production, cell cycle arrest, apoptotic rate and protein expression through WST-1 assay, wound healing assay, flow cytometry and western blotting. Furthermore, the expression levels of proteins, including NOTCH, involved in the pathogenesis of solid cancers, such as melanoma, were examined. Overall, the ORT/DTIC combination synergistically promoted cell cycle arrest at the S phase and the apoptosis of WM-266-4 cells. Thus, this combination treatment may serve as a novel therapeutic strategy for treating malignant melanoma.

Aqueous extracts of Sanghuangporus vaninii induce S-phase arrest and apoptosis in human melanoma A375 cells.

Sanghuangporus vaninii, also called 'Sanghuang' mushroom in Chinese, has various medicinal uses, but its effects on human melanoma cells have not been reported. The present study investigated the inhibitory ability and potential anticancer mechanism of the aqueous extracts of S. vaninii (SH). The results revealed that SH inhibited the proliferation of A375 human melanoma cells in a dose-dependent manner, and flow cytometry analysis suggested that SH induced A375 cell cycle arrest at S phase and apoptosis. Reverse transcription-quantitative PCR, western blotting and immunofluorescence analyses indicated that SH induced S-phase arrest by upregulating p21 expression, and p21 inhibited the expression of cyclin-cyclin-dependent kinases complexes at both the RNA and protein levels. In addition, SH induced apoptosis of A375 cells by inhibiting the expression levels of the anti-apoptosis gene Bcl-2. Therefore, the results suggested that SH may be a potential candidate for the treatment of human melanoma, thus providing new ideas for developing drugs that target melanoma.

Coronavirus Porcine Epidemic Diarrhea Virus Nucleocapsid Protein Interacts with p53 To Induce Cell Cycle Arrest in S-Phase and Promotes Viral Replication.

Subversion of the host cell cycle to facilitate viral replication is a common feature of coronavirus infections. Coronavirus nucleocapsid (N) protein can modulate the host cell cycle, but the mechanistic details remain largely unknown. Here, we investigated the effects of manipulation of porcine epidemic diarrhea virus (PEDV) N protein on the cell cycle and the influence on viral replication. Results indicated that PEDV N induced Vero E6 cell cycle arrest at S-phase, which promoted viral replication (P < 0.05). S-phase arrest was dependent on the N protein nuclear localization signal S71NWHFYYLGTGPHADLRYRT90 and the interaction between N protein and p53. In the nucleus, the binding of N protein to p53 maintained consistently high-level expression of p53, which activated the p53-DREAM pathway. The key domain of the N protein interacting with p53 was revealed to be S171RGNSQNRGNNQGRGASQNRGGNN194 (NS171-N194), in which G183RG185 are core residues. NS171-N194 and G183RG185 were essential for N-induced S-phase arrest. Moreover, small molecular drugs targeting the NS171-N194 domain of the PEDV N protein were screened through molecular docking. Hyperoside could antagonize N protein-induced S-phase arrest by interfering with interaction between N protein and p53 and inhibit viral replication (P < 0.05). The above-described experiments were also validated in porcine intestinal cells, and data were in line with results in Vero E6 cells. Therefore, these results reveal the PEDV N protein interacts with p53 to activate the p53-DREAM pathway, and subsequently induces S-phase arrest to create a favorable environment for virus replication. These findings provide new insight into the PEDV-host interaction and the design of novel antiviral strategies against PEDV. IMPORTANCE Many viruses subvert the host cell cycle to create a cellular environment that promotes viral growth. PEDV, an emerging and reemerging coronavirus, has led to substantial economic loss in the global swine industry. Our study is the first to demonstrate that PEDV N-induced cell cycle arrest during the S-phase promotes viral replication. We identified a novel mechanism of PEDV N-induced S-phase arrest, where the binding of PEDV N protein to p53 maintains consistently high levels of p53 expression in the nucleus to mediate S-phase arrest by activating the p53-DREAM pathway. Furthermore, a small molecular compound, hyperoside, targeted the PEDV N protein, interfering with the interaction between the N protein and p53 and, importantly, inhibited PEDV replication by antagonizing cell cycle arrest. This study reveals a new mechanism of PEDV-host interaction and also provides a novel antiviral strategy for PEDV. These data provide a foundation for further research into coronavirus-host interactions.

A novel small-molecule fatty acid synthase inhibitor with antitumor activity by cell cycle arrest and cell division inhibition.

Fatty acid synthase (FASN), the key enzyme in de novo lipogenesis, is an attractive therapeutic target for diseases characterized by excessive lipid accumulation. Many FASN inhibitors have failed in the clinical trial phase, largely because of poor solubility and safety. In this study, we generated a novel small-molecule FASN inhibitor by structure-based virtual screening. PFI09, the lead compound, is easy to synthesize, and inhibits the lipid synthesis in OP9 mammalian cell line and Caenorhabditis elegans as well as the proliferation of several cancer cell lines via the blockade of FASN. Mechanistic investigations show that PFI09 induces S-phase arrest, cell division reduction and apoptosis. We also develop a chemically stable analog of PFI09, MFI03, which reduces the proliferation of PC3 tumor cells both in vitro and in vivo, without toxicity to mice. In summary, our data suggest that MFI03 is an effective FASN inhibitor and a promising antineoplastic drug candidate.

Chloroform Fraction of Methanolic Extract of Seeds of Annona muricata Induce S Phase Arrest and ROS Dependent Caspase Activated Mitochondria-Mediated Apoptosis in Triple-Negative Breast Cancer.

BACKGROUND: Triple Negative Breast Cancers (TNBCs) have high morbidity and shorter survival rate in the population. These types of cancers have high aggressiveness, lymphatic invasion, and absence of receptors. The treatment options for these types of cancers are also scarce. Several studies have been conducted to investigate the effectiveness of seeds of Annona muricata for its anti-cancer activities in various cancer cell lines, such as lung A549, breast MCF7, colon HT-29, oral KB, and human hepatoma cell lines. But works related to its anti-cancer effect and mechanism of action in TNBCs have not been elucidated. OBJECTIVE: The present study was undertaken to evaluate the in vitro, in vivo, and in silico anti-cancer potential of chloroform fraction of methanolic extract of seeds of Annona muricata (CMAM) against TNBC along with elucidation of its mechanistic pathway. METHODS: In vitro cytotoxicity- and antiproliferative- studies in three triple-negative breast cancer cell lines were conducted using the MTT and SRB assays, respectively. The mechanism through which CMAM exerts its pharmacological effect was elucidated in vitro employing cell morphological assessment studies using Acridine Orange/Ethidium Bromide (AO/EB), intracellular reactive oxygen species assay, DNA fragmentation assay, agarose gel electrophoresis, terminal deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) assay, cell cycle analysis, annexin binding assay, and caspase-activated mitochondria-mediated apoptotic assays using western blot. In vivo evaluation in 4T1 induced murine mammary tumor model was also conducted. Phytoconstituents in CMAM were analyzed using liquid chromatography mass spectroscopy. In silico binding studies with various annonaceous acetogenins against BCL-2 and cyclin E were performed. RESULTS: Cytotoxicity studies in MDA-MD-231, 4TI, and BT-549 revealed the IC50 value of CMAM to be 2.5±0.14, 4.8±0.3, and 4.5±0.16µg/mL, respectively. Anti-proliferative studies in 4T1, MDA-MB-231, and BT- 549 revealed the GI50 values to be 0.128+0.03, 18.03+0.20, 0.95+0.04µg/mL, respectively. CMAM exhibited its cytotoxicity through the lysis of cell membrane, ROS dependent caspase-activated mitochondria-mediated apoptosis, and arresting the S phase of the cell cycle. In vivo evaluation also supported the tumoricidal property of CMAM, as evidenced by a reduction in tumor volume and serum biomarkers. Histopathologically, there was a marked reduction in cellularity, nuclear chromatin condensation, and a few normal cells in the group treated with CMAM at a dose of 31mg/Kg. Phytoconstituent evaluation has revealed the presence of annonaceous acetogenins in CMAM. Among the various annonaceous acetogenins, muricatacin alone showed lipophilicity and binding affinity towards BCL-2 and cyclin E1. CONCLUSION: The current study shows the effectiveness of CMAM against TNBC both in vitro and in vivo. This anticancerous effect of CMAM could be by virtue of its ROS dependent caspase-activated mitochondriamediated apoptosis and the S-phase arrest of the cell cycle in the TNBCs. Our results indicate that the presence of annonaceous acetogenins, especially muricatacin, could be contributing to this anticancerous effect of CMAM. Thus, muricatacin could be a potential candidate for the targeted therapy of TNBCs.

Inhibitory Effect and Mechanism of Jingulian Capsule on Human Breast Cancer MDA-MB-231 Cells / 中国实验方剂学杂志

Objective:To observe effect of Jingulian capsule on the proliferation of human breast cancer MDA-MB-231 cells and investigate its action mechanism against triple negative breast cancer （TNBC）. Method:The ingredients of Jingulian capsule were identified by ultra-performance liquid chromatography-tandem mass spectrometry（UPLC-MS/MS）. The inhibitory effect of Jingulian capsule at different doses （0.125，0.25，0.5，1，and 2 g·L^-1） against the proliferation of MDA-MB-231 cells were detected by methyl thiazolyl tetrazolium （MTT） assay. After treatment for 24 h， the morphological changes in nuclear apoptosis of MDA-MB-231 cells were detected by Hoechst 33258 staining. The effect of different concentrations of Jingulian capsule on the apoptosis and cycle of MDA-MB-231 cells after different treatment time were determined by flow cytometry. The protein expression levels of Poly-ADP-ribose polymeras （PARP）， proto-oncogene c-Myc， cyclin B₁， and phosphorylated extracellular signal-regulated kinase （p-ERK） in each group were assayed by Western blot. Result:A total of 113 compounds in Jingulian capsule were identified by UPLC-MS/MS. As revealed by MTT assay，compared with blank group，Jingulian capsule （0.125，0.25，0.5，1，2 g·L^-1） significantly inhibited viability of MDA-MB-231 cells （<italic>P</italic><0.01）， with the half maximal inhibitory concentration （ IC₅₀） of（0.13±0.02）g·L^-1. According to flow cytometry，compared with the blank group，Jingulian capsule at 1 g·L^-1 significantly induced the apoptosis of MDA-MB-231 cells （<italic>P</italic><0.05）and Jingulian capsule at 0.5， 1 g·L^-1 obviously increased the number of MDA-MB-231 cells in S phase （<italic>P</italic><0.05，<italic>P</italic><0.01）. The results of Western blotting demonstrated that the protein expression levels of PARP，c-Myc，and cyclin B₁ in 0.5， 1 g·L^-1Jingulian capsule groups were remarkably down-regulated as compared with those in the blank group（<italic>P</italic><0.01）， and the protein expression level of p-ERK in 1 g·L^-1Jingulian capsule group was also down-regulated （<italic>P</italic><0.01）. Conclusion:Jingulian capsule is able to inhibit the proliferation of MDA-MB-231 cells，induce S phase cell cycle arrest， and promote their apoptosis， which may be related to the inactivation of the MAPK signaling pathway.

Transcriptome Profiling and Cytological Assessments for Identifying Regulatory Pathways Associated With Diorcinol N-Induced Autophagy in A3 Cells.

Fungal secondary metabolites serve as a rich resource for exploring lead compounds with medicinal importance. Diorcinol N (DN), a fungal secondary metabolite isolated from an endophytic fungus, Arthrinium arundinis, exhibits robust anticancer activity. However, the anticancer mechanism of DN remains unclear. In this study, we examined the growth-inhibitory effect of DN on different human cancer cell lines. We found that DN decreased the viability of A3 T-cell leukemia cells in a time- and concentration-dependent manner. Transcriptome analysis indicated that DN modulated the transcriptome of A3 cells. In total, 9,340 differentially expressed genes were found, among which 4,378 downregulated genes and 4,962 upregulated genes were mainly involved in autophagy, cell cycle, and DNA replication. Furthermore, we demonstrated that DN induced autophagy, cell cycle arrest in the G1/S phase, and downregulated the expression of autophagy- and cell cycle-related genes in A3 cells. By labeling A3 cells with acridine orange/ethidium bromide, Hoechst 33,258, and monodansylcadaverine and via transmission electron microscopy, we found that DN increased plasma membrane permeability, structural disorganization, vacuolation, and autophagosome formation. Our study provides evidence for the mechanism of anticancer activity of DN in T-cell leukemia (A3) cells and demonstrates the promise of DN as a lead or even candidate molecule for the treatment of acute lymphoblastic leukemia.

Small molecule 2,3-DCPE induces S phase arrest by activating the ATM/ATR-Chk1-Cdc25A signaling pathway in DLD-1 colon cancer cells.

In our previous study, it was reported that 2[[3-(2,3-dichlorophenoxy)propyl]amino]ethanol (2,3-DCPE) induces apoptosis and cell cycle arrest. The current study aimed to investigate the molecular mechanism involved in 2,3-DCPE-induced S phase arrest. The results demonstrated that 2,3-DCPE upregulated phosphorylated (p-)H2A histone family member X, a biomarker of DNA damage, in the DLD-1 colon cancer cell line. Western blotting revealed that 2,3-DCPE increased the checkpoint kinase (Chk)1 (Ser317 and Ser345) level and decreased the expression of M-phase inducer phosphatase 1 (Cdc25A) in a time-dependent manner. Subsequently, the results demonstrated that the ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia and Rad3-related (ATR) inhibitors wortmannin and caffeine had no effect on the cell cycle; however, the inhibitors partially abrogated 2,3-DCPE-induced S phase arrest. Flow cytometry assays revealed that caffeine (2 mM) reduced the proportion of S phase cells from 83 to 39.6% and that wortmannin (500 nM) reduced the proportion of S phase cells from 83 to 48.2%. Furthermore, wortmannin and caffeine inhibited the 2,3-DCPE-mediated phosphorylation of Chk1 and the degradation of Cdc25A. However, these ATM/ATR inhibitors had limited effect on 2,3-DCPE-induced apoptosis. Taken together, the data of the current study indicated that 2,3-DCPE caused DNA damage in colon cancer cells and that 2,3-DCPE-induced S phase arrest was associated with the activation of the ATM/ATR-Chk1-Cdc25A pathway.

Water-Extracted Ganoderma lucidum Induces Apoptosis and S-Phase Arrest via Cyclin-CDK2 Pathway in Glioblastoma Cells.

Glioblastoma is one of the most common and most aggressive brain cancers. The current treatment is mainly surgery, chemotherapy, and radiation therapy, but the results are not satisfactory. Ganoderma lucidum (G. lucidum), also called "Lingzhi", is a medicinal mushroom that has been used as a therapeutic agent for the treatment of numerous diseases, including cancer. However, whether it is effective for treating cancer is still unclear. In the present study, the anti-tumor effect of a water extract of G. lucidum was investigated using brain tumor cells. We used an analysis of cell viability, flow cytometry, the IncuCyte live-cell analysis system, and Western blotting to study its effects. The water extract from G. lucidum inhibited cell proliferation in a dose- and time-dependent manner, and it induced mitochondria-mediated apoptosis and cell cycle arrest at S phase via the cyclin-CDK2 pathway in human brain tumor cells. In addition, the G. lucidum extract significantly inhibited cell migration and mesenchymal marker expression based on the IncuCyte live-cell assay and qRT-PCR analysis. In summary, these anti-tumor effects in brain tumor cells suggest that G. lucidum may be useful for treating brain tumors.

In vitro stress response induced by sulfur mustard in lung fibroblasts NHLF and human pulmonary epithelial cells A-549.

Sulfur mustard [bis(2-chloroethyl) sulfide; SM] is a highly poisonous chemical warfare agent. The mechanism of its cytotoxicity affects several pathways, which cause cell damage or death. The main organ affected in case of exposure to both aerosol and vapor is lungs. The present study focuses on time- and concentration-dependent changes in human lung fibroblasts NHLF and lung epithelial cell line A-549. The cells were treated with SM at the concentrations of 5, 10 and 100 µM and signs of stress response were evaluated during 1-72 h post-treatment. Parameters for testing included cell viability and morphology, loss of transmembrane mitochondrial potential, apoptosis, oxidative stress, changes in the cell cycle, and ATM kinase activation. The cytotoxic effect of SM resulted in a time-dependent decrease in viability of A-459 associated with apoptosis more markedly than in NHLF. We did not observe any generation of reactive oxygen species by SM. SM at concentrations of 5 and 10 µM induced the S-phase cell cycle arrest at both cell lines. On the other hand, 100 µM caused nonspecific cell cycle arrest. ATM kinase was activated transiently. The results indicate that NHLF cells are less prone to toxic damage by SM in case of cell viability, apoptosis and loss of transmembrane mitochondrial potential. The analysis provides a time-related cytotoxic profile of A-549 and NHLF cells for further investigation into the prevention of SM toxic effects and their potential treatment.

Synthesis and antitumor activity of α,ß-unsaturated carbonyl moiety- containing oleanolic acid derivatives targeting PI3K/AKT/mTOR signaling pathway.

Oleanolic acid (OA) and its semi-synthetic derivatives have been reported to have a wide range of biological activities. The introduction of electrophilic Michael acceptor group can increase the reactivity of OA to cellular targets and thus improve the anti-tumor activity. In this work, a series of novel α,ß-unsaturated carbonyl derivatives of OA were designed and synthesized. Their in vitro cytotoxic activity against MCF-7, HepG2 and HeLa cells were tested. Most derivatives exhibited improved cell growth inhibitory activity, especially for 3d with an IC50 of 0.77 µM in MCF-7 cells. Moreover, 3d inhibited the migration of MCF-7 and HeLa cells at the concentration of 4 µM. Flow cytometric analysis revealed that 3d induced cell apoptosis and S phase arrest in a concentration-dependent manner. Western blotting experiment demonstrated that 3d inhibited the phosphorylation of AKT and mTOR. These results suggest that this series of OA derivatives bearing exocyclic methylene ketone pharmacophore are promising anticancer agents as potential PI3K/AKT/mTOR pathway inhibitors.

Dihydroorotate Dehydrogenase Inhibitors Promote Cell Cycle Arrest and Disrupt Mitochondria Bioenergetics in Ramos Cells.

BACKGROUND: The re-emerging of targeting Dihydroorotate Dehydrogenase (DHODH) in cancer treatment particularly Acute Myelogenous Leukemia (AML) has corroborated the substantial role of DHODH in cancer and received the attention of many pharmaceutical industries. OBJECTIVE: The effects of Brequinar Sodium (BQR) and 4SC-101 on lymphoblastoid cell lines were investigated. METHODS: DHODH expression and cell proliferation inhibition of lymphoblastoid and lymphoma cell lines were analyzed using Western blot analysis and XTT assay, respectively. JC-1 probe and ATP biochemiluminescence kit were used to evaluate the mitochondrial membrane potential and ATP generation in these cell lines. Furthermore, we explored the cell cycle progression using Muse™ Cell Cycle Kit. RESULTS: Ramos, SUDHL-1 and RPMI-1788 cells are fast-growing cells with equal expression of DHODH enzyme and sensitivity to DHODH inhibitors that showed that the inhibition of DHODH was not cancer-specific. In ATP depletion assay, the non-cancerous RPMI-1788 cells showed only a minor ATP reduction compared to Ramos and SUDHL-1 (cancer) cells. In the mechanistic impact of DHODH inhibitors on non-cancerous vs cancerous cells, the mitochondrial membrane potential assay revealed that significant depolarization and cytochrome c release occurred with DHODH inhibitors treatment in Ramos but not in the RPMI-1788 cells, indicating a different mechanism of proliferation inhibition in normal cells. CONCLUSION: The findings of this study provide evidence that DHODH inhibitors perturb the proliferation of non-cancerous cells via a distinct mechanism compared to cancerous cells. These results may lead to strategies for overcoming the impact on non-cancerous cells during treatment with DHODH inhibitors, leading to a better therapeutic window in patients.

The third-generation retinoid adapalene triggered DNA damage to induce S-phase arrest in HaCat cells.

Epidermal proliferative diseases consisted of a series of common skin diseases, most of which were recurrent chronic skin diseases, and had greatly negative influence on the life quality of patient. Retinoids exhibited vital roles in the treatment of many skin diseases. Our recent study demonstrated that adapalene significantly inhibited the growth of HaCat cells, and the inhibitory activity was stronger than other retinoids, such as all-trans-retinoic acid, acitretin, isotretinoin, tazarotene, and bexarotene. Further study showed that adapalene suppressed the colony formation of HaCat cells, and it dramatically triggered S-phase arrest and apoptosis, rather than G1 phase arrest which was reported in other retinoids in several studies. Additionally, adapalene treatment greatly upregulated the protein expression of DNA damage marker γ-H2AX, which was in accord with the results of the elongation of tail moment by comet electrophoresis analysis. Moreover, DNA damage was triggered and DNA repair was suppressed synchronously with adapalene treatment, which accounted for the mechanism of S-phase arrest induced by adapalene. In summary, our recent work demonstrated that adapalene showed strong anti-proliferation activity in HaCat cells and could be an alternative agent for the epidermal proliferative disease.

Anticancer Activity of Platinum (II) Complex with 2-Benzoylpyridine by Induction of DNA Damage, S-Phase Arrest, and Apoptosis.

OBJECTIVE: To overcome the disadvantages of cisplatin, numerous platinum (Pt) complexes have been prepared. However, the anticancer activity and mechanism of Pt(II) complexed with 2-benzoylpyridine [Pt(II)- Bpy]: [PtCl2(DMSO)L] (DMSO = dimethyl sulfoxide, L = 2-benzoylpyridine) in cancer cells remain unknown. METHODS: Pt(II)-Bpy was synthesized and characterized by spectrum analysis. Its anticancer activity and underlying mechanisms were demonstrated at the cellular, molecular, and in vivo levels. RESULTS: Pt(II)-Bpy inhibited tumor cell growth, especially HepG2 human liver cancer cells, with a halfmaximal inhibitory concentration of 9.8±0.5µM, but with low toxicity in HL-7702 normal liver cells. Pt(II)- Bpy induced DNA damage, which was demonstrated through a marked increase in the expression of cleavedpoly (ADP ribose) polymerase (PARP) and gamma-H2A histone family member X and a decrease in PARP expression. The interaction of Pt(II)-Bpy with DNA at the molecular level was most likely through an intercalation mechanism, which might be evidence of DNA damage. Pt(II)-Bpy initiated cell cycle arrest at the S phase in HepG2 cells. It also caused severe loss of the mitochondrial membrane potential; a decrease in the expression of caspase-9 and caspase-3; an increase in reactive oxygen species levels; the release of cytochrome c and apoptotic protease activation factor; and the activation of caspase-9 and caspase-3 in HepG2 cells, which in turn resulted in apoptosis. Meanwhile, changes in p53 and related proteins were observed including the upregulation of p53, the phosphorylation of p53, p21, B-cell lymphoma-2-associated X protein, and NOXA; and the downregulation of B-cell lymphoma 2. Moreover, Pt(II)-Bpy displayed marked inhibitory effects on tumor growth in the HepG2 nude mouse model. CONCLUSION: Pt(II)-Bpy is a potential candidate for cancer chemotherapy.