Aroylhydrazone Diorganotin Complexes Causes DNA Damage and Apoptotic Cell Death: From Chemical Synthesis to Biochemical Effects.

Under microwave irradiation, eighteen new aroylhydrazone diorganotin complexes (1a-9b) were produced through the reaction of aroylhydrazine, 2-ketobutyric acid, and the corresponding diorganotin. Fourier transform infrared spectroscopy, 1H, 13C, and 119Sn nuclear magnetic resonance spectroscopies, high-resolution mass spectroscopy, X-ray crystallography, and thermogravimetric analysis (TGA) were performed to characterize the complexes. The in vitro anticancer activity for complexes were assessed using a CCK-8 assay on human cancer cells of HepG2, NCI-H460, and MCF-7. Complex 4b revealed more intensive anticancer activity against MCF-7 cells than the other complexes and cisplatin. Flow cytometry analysis and transmission electron microscope observation demonstrated that complex 4b mediated cell apoptosis of MCF-7 cells and arrested cell cycle in S phase. Western blotting analysis showed that 4b induced DNA damage in MCF-7 cells and led to apoptosis by the ATM-CHK2-p53 pathway. The single cell gel electrophoreses assay results showed that 4b induced DNA damage. The DNA binding activity of 4b was studied by UV-Visible absorption spectrometry, fluorescence competitive, viscosity measurements, gel electrophoresis, and molecular docking, and the results show that 4b can be well embedded in the groove and cleave DNA.

Tanshinone IIA derivatives induced S-phase arrest through stabilizing c-myc G-quadruplex DNA to regulate ROS-mediated PI3K/Akt/mTOR pathway.

Herein, a derivate from tanshinone IIA, 1,6,6-trimethyl-11-phenyl-7,8,9,10-tetrahydro-6H-furo[2',3':1,2]phenanthro[3,4-d]imidazole (TA25), has been synthesized and investigated as potential inhibitor against the proliferation, migration and invasion of lung cancer cells. MTT assay and cell colony formation assay results showed that TA25 exhibits acceptable inhibitory effect against the proliferation of lung cancer A549 cells, and the value of IC50 was about 17.9 µM. This result was further confirmed by the inhibition of TA25 against the growth of xenograft lung cancer cells on zebrafish bearing tumor (A549 lung cancer cells). The results of wound-healing assay and FITC-gelatin invasion assay displayed that TA25 could inhibit the migration and invasion of lung cancer A549 cells. Moreover, the studies on the binding properties of TA25 interact with c-myc G-quadruplex DNA suggested that TA25 can bind in the G-quarter plane formed from G7, G11, G16 and G20 with c-myc G-quadruplex DNA through π-π stacking. Further study of the potential anti-cancer mechanism indicated that TA25 can induce S-phase arrest in lung cancer A549 cells, and this phenomenon resulted from the promotion of the production of reactive oxygen species and DNA damage in A549 cells under the action of TA25. Further research revealed that TA25 could inhibit the PI3K/Akt/mTOR signal pathway and increase the expression of p53 protein. Overall, TA25 can be developed into a promising inhibitor against the proliferation, migration and invasion of lung cancer cells and has potential clinical application in the near future.

A regulatory phosphorylation site on Mec1 controls chromatin occupancy of RNA polymerases during replication stress.

Upon replication stress, budding yeast checkpoint kinase Mec1ATR triggers the downregulation of transcription, thereby reducing the level of RNA polymerase (RNAP) on chromatin to facilitate replication fork progression. Here, we identify a hydroxyurea-induced phosphorylation site on Mec1, Mec1-S1991, that contributes to the eviction of RNAPII and RNAPIII during replication stress. The expression of the non-phosphorylatable mec1-S1991A mutant reduces replication fork progression genome-wide and compromises survival on hydroxyurea. This defect can be suppressed by destabilizing chromatin-bound RNAPII through a TAP fusion to its Rpb3 subunit, suggesting that lethality in mec1-S1991A mutants arises from replication-transcription conflicts. Coincident with a failure to repress gene expression on hydroxyurea in mec1-S1991A cells, highly transcribed genes such as GAL1 remain bound at nuclear pores. Consistently, we find that nuclear pore proteins and factors controlling RNAPII and RNAPIII are phosphorylated in a Mec1-dependent manner on hydroxyurea. Moreover, we show that Mec1 kinase also contributes to reduced RNAPII occupancy on chromatin during an unperturbed S phase by promoting degradation of the Rpb1 subunit.

Loss of haspin suppresses cancer cell proliferation by interfering with cell cycle progression at multiple stages.

Our recent studies have shown that haspin, a protein kinase imperative for mitosis, is engaged in the interphase progression of HeLa and U2OS cancer cells. In this investigation, we employed the Fucci reporter system and time-lapse imaging to examine the impact of haspin gene silencing on cell cycle progressions at a single-cell level. We found that the loss of haspin induced multiple cell cycle defects. Specifically, the S/G2 duration was greatly prolonged by haspin gene depletion or inhibition in synchronous HeLa cells. Haspin gene depletion in asynchronous HeLa and U2OS cells led to a similarly protracted S/G2 phase, followed by mitotic cell death or postmitotic G1 arrest. In addition, haspin deficiency resulted in robust induction of the p21CIP1/WAF1 checkpoint protein, a target of the p53 activation. Also, co-depleting haspin with either p21 or p53 could rescue U2OS cells from postmitotic G1 arrest and partially restore their proliferation. These results substantiate the haspin's capacity to regulate interphase and mitotic progression, offering a broader antiproliferative potential of haspin loss in cancer cells.

Hydroxyurea-The Good, the Bad and the Ugly.

Hydroxyurea (HU) is mostly referred to as an inhibitor of ribonucleotide reductase (RNR) and as the agent that is commonly used to arrest cells in the S-phase of the cycle by inducing replication stress. It is a well-known and widely used drug, one which has proved to be effective in treating chronic myeloproliferative disorders and which is considered a staple agent in sickle anemia therapy and-recently-a promising factor in preventing cognitive decline in Alzheimer's disease. The reversibility of HU-induced replication inhibition also makes it a common laboratory ingredient used to synchronize cell cycles. On the other hand, prolonged treatment or higher dosage of hydroxyurea causes cell death due to accumulation of DNA damage and oxidative stress. Hydroxyurea treatments are also still far from perfect and it has been suggested that it facilitates skin cancer progression. Also, recent studies have shown that hydroxyurea may affect a larger number of enzymes due to its less specific interaction mechanism, which may contribute to further as-yet unspecified factors affecting cell response. In this review, we examine the actual state of knowledge about hydroxyurea and the mechanisms behind its cytotoxic effects. The practical applications of the recent findings may prove to enhance the already existing use of the drug in new and promising ways.

Brusatol inhibits laryngeal cancer cell proliferation and metastasis via abrogating JAK2/STAT3 signaling mediated epithelial-mesenchymal transition.

AIMS: This study aimed at investigating the role of Brusatol (BR) on human laryngeal squamous carcinoma cell (Hep-2) to study its underlying mechanism through in vitro and in vivo approaches. MATERIALS AND METHOD: In the present research, we employed various cell-based assays, such as cell proliferation, apoptosis, cell cycle assessment, migration and invasion assays were used to examine the anti-tumor effect of BR on Hep-2 cells. Immunohistochemistry (IHC), qRT-PCR and Western blotting were performed to study the underlying molecular mechanisms. To validate our in vitro findings we used a subcutaneous tumor-bearing model of Balb/c mice with Hep-2 cells of laryngeal carcinoma (LC) to study the inhibitory effect of BR on Hep-2 cells in vivo. KEY FINDINGS: The results indicated that BR markedly inhibited the viability, migration and invasion capacity of Hep-2 cells, with no significant toxic effect on normal Human bronchial epithelial cell line (BEAS-2B). Also, BR induced cellular apoptosis by blocking the cells in S phase to suppress cell proliferation. Immunohistochemistry results revealed that BR inhibited the protein expression levels of epithelial-mesenchymal transition (EMT)-related markers. Mechanistically, western blotting results exhibited that BR could suppress the protein expression of both JAK2/STAT3 and their phosphorylation levels. Our in vivo experiments further validated the anti-tumor effect of BR on Hep-2 cells in vitro, where BR suppressed the growth of xenograft laryngeal tumor without apparent toxicity. SIGNIFICANCE: The present study highlights the anti-LC effect of BR by possibly abrogating JAK2/STAT3 signaling mediated EMT process. BR may be a promising therapeutic candidate for the treatment of LC.

The synergistic antitumor effect of combination therapy with a MEK inhibitor and YAP inhibitor on pERK-positive neuroblastoma.

BACKGROUND: We previously reported the in vitro and in vivo antitumor effects of trametinib, a MEK inhibitor, on neuroblastoma with MAPK pathway mutations. As we observed eventual resistance to trametinib in our previous study, we evaluated the combination therapy of CA3, a YAP inhibitor, with trametinib, based on a recent report suggesting the potential involvement of YAP in the mechanism underlying the resistance to trametinib in neuroblastoma. METHODS: SK-N-AS cells (a neuroblastoma cell line harboring RAS mutation) were treated with CA3 in vitro and subjected to a viability assay, immunocytochemistry and flow cytometry. Next, we analyzed the in vitro combination effect of CA3 and trametinib using the CompuSyn software program. Finally, we administered CA3, trametinib or both to SK-N-AS xenograft mice for 10 weeks to analyze the combination effect. RESULTS: CA3 inhibited cell proliferation by both cell cycle arrest and apoptosis in vitro. Combination of CA3 and trametinib induced a significant synergistic effect in vitro (Combination Index <1). Regarding the in vivo experiment, combination therapy suppressed tumor growth, and 100% of mice in the combination therapy group survived, whereas the survival rates were 0% in the CA3 group and 33% in the trametinib group. However, despite this promising survival rate in the combination group, the tumors gradually grew after seven weeks with MAPK reactivation. CONCLUSION: Our results indicated that CA3 and trametinib exerted synergistic antitumor effects on neuroblastoma in vitro and in vivo, and CA3 may be a viable option for concomitant drug therapy with trametinib, since it suppressed the resistance to trametinib. However, this combination effect was not sufficient to achieve complete remission. Therefore, we need to adjust the protocol to obtain a better outcome by determining the mechanism underlying regrowth in the future.

Hydroxyphenyl Butanone Induces Cell Cycle Arrest through Inhibition of GSK3ß in Colorectal Cancer.

BACKGROUND: Colorectal cancer (CRC) is among the top three gastrointestinal malignancy in morbidity and mortality. The abnormal activation of Wnt/ß-catenin pathway is considered to be a key factor in the occurrence and development of CRC. Novel inhibitor discovery against key factor in WNT pathway is important for CRC treatment and prevention. METHODS: Cell proliferation was detected after hydroxyphenyl butanone treatment in human colorectal cancer HCT116, LOVO, and normal colonic epithelial NCM460 cells. Colony formation, cell invasion ability, and cell cycle were detected with and without GSK-3ß knockdown. RESULTS: Hydroxyphenyl butanone induces cycle arresting on G1-S phase of colorectal cancer cell line through GSK3ß in Wnt/ß-catenin pathway and inhibits malignant biological manifestations of cell proliferation, colony formation, and invasion. The inhibition in the high concentration group is stronger than that in the low concentration group, and the antitumor effect is different for different tumor cells. Under the same concentration of natural hydroxyphenyl butanone, the inhibition on normal colonic epithelial cells is significantly lower than that on tumor cells. The natural hydroxyphenyl butanone with medium and low concentration could promote the proliferation of normal colonic epithelial cells. CONCLUSION: This study illustrated natural hydroxyphenyl butanone as new inhibitor of GSK3ß and revealed the mechanisms underlying the inhibitory effects in colorectal cancer.

Local ablation of gastric cancer by reconstituted apolipoprotein B lipoparticles carrying epigenetic drugs.

Epigenetic inhibitors have shown anticancer effects. Combination chemotherapy with epigenetic inhibitors has shown high effectiveness in gastric cancer clinical trials, but severe side effect and local progression are the causes of treatment failure. Therefore, we sought to develop an acidity-sensitive drug delivery system to release drugs locally to diminish unfavorable outcome of gastric cancer. In this study, we showed that, as compared with single agents, combination treatment with the demethylating agent 5'-aza-2'-deoxycytidine and HDAC inhibitors Trichostatin A or LBH589 decreased cell survival, blocked cell cycle by reducing number of S-phase cells and expression of cyclins, increased cell apoptosis by inducing expression of Bim and cleaved Caspase 3, and reexpressed tumor suppressor genes more effectively in MGCC3I cells. As a carrier, reconstituted apolipoprotein B lipoparticles (rABLs) could release drugs in acidic environments. Orally administrated embedded drugs not only showed inhibitory effects on gastric tumor growth in a syngeneic orthotopic mouse model, but also reduced the hepatic and renal toxicity. In conclusion, we have established rABL-based nanoparticles embedded epigenetic inhibitors for local treatment of gastric cancer, which have good therapeutic effects but do not cause severe side effects.

CAP1 binds and activates adenylyl cyclase in mammalian cells.

CAP1 (Cyclase-Associated Protein 1) is highly conserved in evolution. Originally identified in yeast as a bifunctional protein involved in Ras-adenylyl cyclase and F-actin dynamics regulation, the adenylyl cyclase component seems to be lost in mammalian cells. Prompted by our recent identification of the Ras-like small GTPase Rap1 as a GTP-independent but geranylgeranyl-specific partner for CAP1, we hypothesized that CAP1-Rap1, similar to CAP-Ras-cyclase in yeast, might play a critical role in cAMP dynamics in mammalian cells. In this study, we report that CAP1 binds and activates mammalian adenylyl cyclase in vitro, modulates cAMP in live cells in a Rap1-dependent manner, and affects cAMP-dependent proliferation. Utilizing deletion and mutagenesis approaches, we mapped the interaction of CAP1-cyclase with CAP's N-terminal domain involving critical leucine residues in the conserved RLE motifs and adenylyl cyclase's conserved catalytic loops (e.g., C1a and/or C2a). When combined with a FRET-based cAMP sensor, CAP1 overexpression-knockdown strategies, and the use of constitutively active and negative regulators of Rap1, our studies highlight a critical role for CAP1-Rap1 in adenylyl cyclase regulation in live cells. Similarly, we show that CAP1 modulation significantly affected cAMP-mediated proliferation in an RLE motif-dependent manner. The combined study indicates that CAP1-cyclase-Rap1 represents a regulatory unit in cAMP dynamics and biology. Since Rap1 is an established downstream effector of cAMP, we advance the hypothesis that CAP1-cyclase-Rap1 represents a positive feedback loop that might be involved in cAMP microdomain establishment and localized signaling.

Composites of Nucleic Acids and Boron Clusters (C2B10H12) as Functional Nanoparticles for Downregulation of EGFR Oncogene in Cancer Cells.

Epidermal growth factor receptor (EGFR) is one of the most promising molecular targets for anticancer therapy. We used boron clusters as a platform for generation of new materials. For this, functional DNA constructs conjugated with boron clusters (B-ASOs) were developed. These B-ASOs, built from 1,2-dicarba-closo-dodecaborane linked with two anti-EGFR antisense oligonucleotides (ASOs), form with their complementary congeners torus-like nanostructures, as previously shown by atomic force microscope (AFM) and transmission electron cryo-microscopy (cryo-TEM) imaging. In the present work, deepened studies were carried out on B-ASO's properties. In solution, B-ASOs formed four dominant complexes as confirmed by non-denaturing polyacrylamide gel electrophoresis (PAGE). These complexes exhibited increased stability in cell lysate comparing to the non-modified ASO. Fluorescently labeled B-ASOs localized mostly in the cytoplasm and decreased EGFR expression by activating RNase H. Moreover, the B-ASO complexes altered the cancer cell phenotype, decreased cell migration rate, and arrested the cells in the S phase of cell cycle. The 1,2-dicarba-closo-dodecaborane-containing nanostructures did not activate NLRP3 inflammasome in human macrophages. In addition, as shown by inductively coupled plasma mass spectrometry (ICP MS), these nanostructures effectively penetrated the human squamous carcinoma cells (A431), showing their potential applicability as anticancer agents.

Hypericin-mediated photodynamic therapy inhibits growth of colorectal cancer cells via inducing S phase cell cycle arrest and apoptosis.

Colorectal cancer (CRC) is one type of cancer with high morbidity and mortality worldwide. Photodynamic therapy (PDT), a promising new therapeutic approach for cancer, induces tumor damage through photosensitizer-mediated oxidative cytotoxicity. Hypericin is a powerful photosensitizer with pronounced tumor-localizing properties. In this study, we investigated the phototoxic effects of hypericin-mediated PDT (HYP-PDT) in HCT116 and SW620 cells. We validated that HYP-PDT inhibited cell proliferation, triggered intracellular reactive oxygen species generation, induced S phase cell cycle arrest and apoptosis of HCT116 and SW620 cells. Mechanistically, the results of western blot showed that HYP-PDT downregulated CDK2 expression through decreasing the CDC25A protein, which resulted in the decrease of CDK2/Cyclin A complex. Additionally, HYP-PDT induced DNA damage as evidenced by ATM activation and upregulation of p-H2AX. Further investigation showed that HYP-PDT significantly increased Bax expression and decreased Bcl-2 expression, and then, upregulated the expression of cleaved caspase-9, cleaved caspase-3 and cleaved PARP, thereby inducing apoptosis in HCT116 and SW620 cells. In conclusion, our results indicated that the CDC25A/CDK2/Cyclin A pathway and the mitochondrial apoptosis pathway were involved in HYP-PDT induced S phase cell cycle arrest and apoptosis in colorectal cancer cells, which shows HYP could be a probable candidate used for treating colorectal cancer.

Deficiency of NEIL3 Enhances the Chemotherapy Resistance of Prostate Cancer.

Acquired treatment resistance is an important cause of death in prostate cancer, and this study aimed to explore the mechanisms of chemotherapy resistance in prostate cancer. We employed castration-resistant prostate cancer (CRPC), neuroendocrine prostate cancer (NEPC), and chemotherapy-resistant prostate cancer datasets to screen for potential target genes. The Cancer Genome Atlas (TCGA) was used to detect the correlation between the target genes and prognosis and clinical characteristics. Nei endonuclease VIII-like 3 (NEIL3) knockdown cell lines were constructed with RNA interference. Prostate cancer cells were treated with enzalutamide for the androgen deprivation therapy (ADT) model, and with docetaxel and cisplatin for the chemotherapy model. Apoptosis and the cell cycle were examined using flow cytometry. RNA sequencing and western blotting were performed in the knockdown Duke University 145 (DU145) cell line to explore the possible mechanisms. The TCGA dataset demonstrated that high NEIL3 was associated with a high T stage and Gleason score, and indicated a possibility of lymph node metastasis, but a good prognosis. The cell therapy models showed that the loss of NEIL3 could promote the chemotherapy resistance (but not ADT resistance) of prostate cancer (PCa). Flow cytometry revealed that the loss of NEIL3 in PCa could inhibit cell apoptosis and cell cycle arrest under cisplatin treatment. RNA sequencing showed that the knockdown of NEIL3 changes the expression of neuroendocrine-related genes. Further western blotting revealed that the loss of NEIL3 could significantly promote the phosphorylation of ATR serine/threonine kinase (ATR) and ATM serine/threonine kinase (ATM) under chemotherapy, thus initiating downstream pathways related to DNA repair. In summary, the loss of NEIL3 promotes chemotherapy resistance in prostate cancer, and NEIL3 may serve as a diagnostic marker for chemotherapy-resistant patients.

Protective Effect of Degraded Porphyra yezoensis Polysaccharides on the Oxidative Damage of Renal Epithelial Cells and on the Adhesion and Endocytosis of Nanocalcium Oxalate Crystals.

The protective effects of Porphyra yezoensis polysaccharides (PYPs) with molecular weights of 576.2 (PYP1), 105.4 (PYP2), 22.47 (PYP3), and 3.89 kDa (PYP4) on the oxidative damage of human kidney proximal tubular epithelial (HK-2) cells and the differences in adherence and endocytosis of HK-2 cells to calcium oxalate monohydrate crystals before and after protection were investigated. Results showed that PYPs can effectively reduce the oxidative damage of oxalic acid to HK-2 cells. Under the preprotection of PYPs, cell viability increased, cell morphology improved, reactive oxygen species levels decreased, mitochondrial membrane potential increased, S phase cell arrest was inhibited, the cell apoptosis rate decreased, phosphatidylserine exposure reduced, the number of crystals adhered to the cell surface reduced, but the ability of cells to endocytose crystals enhanced. The lower the molecular weight, the better the protective effect of PYP. The results in this article indicated that PYPs can reduce the risk of kidney stone formation by protecting renal epithelial cells from oxidative damage and reducing calcium oxalate crystal adhesion, and PYP4 with the lowest molecular weight may be a potential drug for preventing kidney stone formation.

The capacity of origins to load MCM establishes replication timing patterns.

Loading of the MCM replicative helicase at origins of replication is a highly regulated process that precedes DNA replication in all eukaryotes. The stoichiometry of MCM loaded at origins has been proposed to be a key determinant of when those origins initiate replication during S phase. Nevertheless, the genome-wide regulation of MCM loading stoichiometry and its direct effect on replication timing remain unclear. In order to investigate why some origins load more MCM than others, we perturbed MCM levels in budding yeast cells and, for the first time, directly measured MCM levels and replication timing in the same experiment. Reduction of MCM levels through degradation of Mcm4, one of the six obligate components of the MCM complex, slowed progression through S phase and increased sensitivity to replication stress. Reduction of MCM levels also led to differential loading at origins during G1, revealing origins that are sensitive to reductions in MCM and others that are not. Sensitive origins loaded less MCM under normal conditions and correlated with a weak ability to recruit the origin recognition complex (ORC). Moreover, reduction of MCM loading at specific origins of replication led to a delay in their replication during S phase. In contrast, overexpression of MCM had no effects on cell cycle progression, relative MCM levels at origins, or replication timing, suggesting that, under optimal growth conditions, cellular MCM levels are not limiting for MCM loading. Our results support a model in which the loading capacity of origins is the primary determinant of MCM stoichiometry in wild-type cells, but that stoichiometry is controlled by origins' ability to recruit ORC and compete for MCM when MCM becomes limiting.

RNA polymerase II condensate formation and association with Cajal and histone locus bodies in living human cells.

In eukaryotic nuclei, a number of phase-separated nuclear bodies (NBs) are present. RNA polymerase II (Pol II) is the main player in transcription and forms large condensates in addition to localizing at numerous transcription foci. Cajal bodies (CBs) and histone locus bodies (HLBs) are NBs that are involved in transcriptional and post-transcriptional regulation of small nuclear RNA and histone genes. By live-cell imaging using human HCT116 cells, we here show that Pol II condensates (PCs) nucleated near CBs and HLBs, and the number of PCs increased during S phase concomitantly with the activation period of histone genes. Ternary PC-CB-HLB associates were formed via three pathways: nucleation of PCs and HLBs near CBs, interaction between preformed PC-HLBs with CBs and nucleation of PCs near preformed CB-HLBs. Coilin knockout increased the co-localization rate between PCs and HLBs, whereas the number, nucleation timing and phosphorylation status of PCs remained unchanged. Depletion of PCs did not affect CBs and HLBs. Treatment with 1,6-hexanediol revealed that PCs were more liquid-like than CBs and HLBs. Thus, PCs are dynamic structures often nucleated following the activation of gene clusters associated with other NBs.

Apoptotic mechanism activated by blue light and cisplatinum in cutaneous squamous cell carcinoma cells.

New approaches are being studied for the treatment of skin cancer. It has been reported that light combined with cisplatinum may be effective against skin cancer. In the present study, the effects of specific light radiations and cisplatinum on A431 cutaneous squamous cell carcinoma (cSCC) and HaCaT non­tumorigenic cell lines were investigated. Both cell lines were exposed to blue and red light sources for 3 days prior to cisplatinum treatment. Viability, apoptosis, cell cycle progression and apoptotic­related protein expression levels were investigated. The present results highlighted that combined treatment with blue light and cisplatinum was more effective in reducing cell viability compared with single treatments. Specifically, an increase in the apoptotic rate was observed when the cells were treated with blue light and cisplatinum, as compared to treatment with blue light or cisplatinum alone. Combined treatment with blue light and cisplatinum also caused cell cycle arrest at the S phase. Treatment with cisplatinum following light exposure induced the expression of apoptotic proteins in the A431 and HaCaT cell lines, which tended to follow different apoptotic mechanisms. On the whole, these data indicate that blue light combined with cisplatinum may be a promising treatment for cSCC.

A novel translation inhibitor, mersicarpine, inhibits S-phase progression and induces apoptosis in HL60 cells.

Mersicarpine is an aspidosperma alkaloid isolated from the Kopsia genus of plants. Its intriguing structural features have attracted much attention in synthetic organic chemistry, but no biological activity has been reported. Here, we report the effects of mersicarpine on human leukemia cell line HL60. At concentrations above 30 µm, mersicarpine reversibly arrested cell cycle progression in S-phase. At higher concentrations, it induced not only production of reactive oxygen species, but also apoptosis. Macromolecular synthesis assay revealed that mersicarpine specifically inhibits protein synthesis. These results suggest that mersicarpine is a novel translation inhibitor that induces apoptosis.

Reprogramming of Nucleotide Metabolism Mediates Synergy between Epigenetic Therapy and MAP Kinase Inhibition.

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare but often lethal cancer that is diagnosed at a median age of 24 years. Optimal management of patients is not well defined, and current treatment remains challenging, necessitating the discovery of novel therapeutic approaches. The identification of SMARCA4-inactivating mutations invariably characterizing this type of cancer provided insights facilitating diagnostic and therapeutic measures against this disease. We show here that the BET inhibitor OTX015 acts in synergy with the MEK inhibitor cobimetinib to repress the proliferation of SCCOHT in vivo Notably, this synergy is also observed in some SMARCA4-expressing ovarian adenocarcinoma models intrinsically resistant to BETi. Mass spectrometry, coupled with knockdown of newly found targets such as thymidylate synthase, revealed that the repression of a panel of proteins involved in nucleotide synthesis underlies this synergy both in vitro and in vivo, resulting in reduced pools of nucleotide metabolites and subsequent cell-cycle arrest. Overall, our data indicate that dual treatment with BETi and MEKi represents a rational combination therapy against SCCOHT and potentially additional ovarian cancer subtypes.

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.