Functional Interactions between Herpesvirus Oncoprotein MEQ and Cell Cycle Regulator CDK2

ABSTRACT Marek’s disease virus, an avian alphaherpesvirus, has been used as an excellent model to study herpesvirus oncogenesis. One of its potential oncogenes, MEQ, has been demonstrated to transform a rodent fibroblast cell line, Rat-2, in vitro by inducing morphological transformation and anchorage- and serum-independent growth and by protecting cells from apoptosis induced by tumor necrosis factor alpha, C2-ceramide, UV irradiation, or serum deprivation. In this report, we show that there is a cell cycle-dependent colocalization of MEQ protein and cyclin-dependent kinase 2 (CDK2) in coiled bodies and the nucleolar periphery during the G1/S boundary and early S phase. To our knowledge, this is the first demonstration that CDK2 is found to localize to coiled bodies. Such an in vivo association and possibly subsequent phosphorylation may result in the cytoplasmic translocation of MEQ protein. Indeed, MEQ is expressed in both the nucleus and the cytoplasm during the G1/S boundary and early S phase. In addition, we were able to show in vitro phosphorylation of MEQ by CDKs. We have mapped the CDK phosphorylation site of MEQ to be serine 42, a residue in the proximity of the bZIP domain. An indirect-immunofluorescence study of the MEQ S42D mutant, in which the CDK phosphorylation site was mutated to a charged residue, reveals more prominent cytoplasmic localization. This lends further support to the notion that the translocation of MEQ is regulated by phosphorylation. Furthermore, phosphorylation of MEQ by CDKs drastically reduces the DNA binding activity of MEQ, which may in part account for the lack of retention of MEQ oncoprotein in the nucleus. Interestingly, the localization of CDK2 in coiled bodies and the nucleolar periphery is observed only in MEQ-transformed Rat-2 cells, implicating MEQ in modifying the subcellular localization of CDK2. Taken together, our data suggest that there is a novel reciprocal modulation between the herpesvirus oncoprotein MEQ and CDK2.

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Negative Regulation of Cdc18 DNA Replication Protein by Cdc2

Molecular Biology of the Cell ◽

10.1091/mbc.9.1.63 ◽

1998 ◽

Vol 9 (1) ◽

pp. 63-73 ◽

Cited By ~ 55

Author(s):

Antonia Lopez-Girona ◽

Odile Mondesert ◽

Janet Leatherwood ◽

Paul Russell

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Fission Yeast ◽

Phosphorylation Site ◽

Constitutive Expression ◽

S Phase ◽

Negative Regulation ◽

Chromosomal Dna

Fission yeast Cdc18, a homologue of Cdc6 in budding yeast and metazoans, is periodically expressed during the S phase and required for activation of replication origins. Cdc18 overexpression induces DNA rereplication without mitosis, as does elimination of Cdc2-Cdc13 kinase during G2 phase. These findings suggest that illegitimate activation of origins may be prevented through inhibition of Cdc18 by Cdc2. Consistent with this hypothesis, we report that Cdc18 interacts with Cdc2 in association with Cdc13 and Cig2 B-type cyclins in vivo. Cdc18 is phosphorylated by the associated Cdc2 in vitro. Mutation of a single phosphorylation site, T104A, activates Cdc18 in the rereplication assay. The cdc18-K9 mutation is suppressed by a cig2 mutation, providing genetic evidence that Cdc2-Cig2 kinase inhibits Cdc18. Moreover, constitutive expression of Cig2 prevents rereplication in cells lacking Cdc13. These findings identify Cdc18 as a key target of Cdc2-Cdc13 and Cdc2-Cig2 kinases in the mechanism that limits chromosomal DNA replication to once per cell cycle.

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Differences in cell cycle characteristics among patients with acute nonlymphocytic leukemia

Blood ◽

10.1182/blood.v69.6.1647.bloodjournal6961647 ◽

1987 ◽

Vol 69 (6) ◽

pp. 1647-1653 ◽

Cited By ~ 1

Author(s):

A Raza ◽

Y Maheshwari ◽

HD Preisler

Keyword(s):

Cell Cycle ◽

Tritiated Thymidine ◽

S Phase ◽

Total Cell ◽

Acute Nonlymphocytic Leukemia ◽

Cell Cycle Time ◽

Myeloid Leukemias ◽

History Of

The proliferative characteristics of myeloid leukemias were defined in vivo after intravenous infusions of bromodeoxyuridine (BrdU) in 40 patients. The percentage of S-phase cells obtained from the biopsies (mean, 20%) were significantly higher (P = .00003) than those determined from the bone marrow (BM) aspirates (mean, 9%). The post- BrdU infusion BM aspirates from 40 patients were incubated with tritiated thymidine in vitro. These double-labeled slides were utilized to determine the duration of S-phase (Ts) in myeloblasts and their total cell cycle time (Tc). The Ts varied from four to 49 hours (mean, 19 hours; median, 17 hours). Similarly, there were wide variations in Tc of individual patients ranging from 16 to 292 hours (mean, 93 hours; median, 76 hours). There was no relationship between Tc and the percentage of S-phase cells, but there was a good correlation between Tc and Ts (r = .8). Patients with relapsed acute nonlymphocytic leukemia (ANLL) appeared to have a longer Ts and Tc than those studied at initial diagnosis. A subgroup of patients at either extreme of Tc were identified who demonstrated clinically documented resistance in response to multiple courses of chemotherapy. We conclude that Ts and Tc provide additional biologic information that may be valuable in understanding the variations observed in the natural history of ANLL.

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A CDK-regulated chromatin segregase promoting chromosome replication

10.1101/2020.11.20.390914 ◽

2020 ◽

Author(s):

Erika Chacin ◽

Priyanka Bansal ◽

Karl-Uwe Reusswig ◽

Luis M. Diaz-Santin ◽

Pedro Ortega ◽

...

Keyword(s):

Atp Hydrolysis ◽

Genome Instability ◽

Chromosome Replication ◽

S Phase ◽

Chromatin Dynamics ◽

Replicative Stress ◽

Cdk Phosphorylation ◽

Nucleosome Disassembly

The replication of chromosomes during S phase is critical for cellular and organismal function. Replicative stress can result in genome instability, which is a major driver of cancer. Yet how chromatin is made accessible during eukaryotic DNA synthesis is poorly understood.Here, we report the identification of a novel class of chromatin remodeling enzyme, entirely distinct from classical SNF2-ATPase family remodelers. Yta7 is a AAA+-ATPase that assembles into ~ 1 MDa hexameric complexes capable of segregating histones from DNA. Yta7 chromatin segregase promotes chromosome replication both in vivo and in vitro. Biochemical reconstitution experiments using purified proteins revealed that Yta7’s enzymatic activity is regulated by S phase-forms of Cyclin-Dependent Kinase (S-CDK). S-CDK phosphorylation stimulates ATP hydrolysis by Yta7, promoting nucleosome disassembly and chromatin replication.Our results present a novel mechanism of how cells orchestrate chromatin dynamics in co-ordination with the cell cycle machinery to promote genome duplication during S phase.

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Alterations in the cell cycle of mouse cumulus granulosa cells during expansion and mucification in vivo and in vitro

Reproduction Fertility and Development ◽

10.1071/rd9960935 ◽

1996 ◽

Vol 8 (6) ◽

pp. 935 ◽

Cited By ~ 25

Author(s):

AW Schuetz ◽

DG Whittingham ◽

R Snowden

Keyword(s):

Cell Cycle ◽

Dna Synthesis ◽

Granulosa Cells ◽

Dna Content ◽

Cumulus Cell ◽

Cumulus Cells ◽

Ovarian Follicles ◽

S Phase

The cell cycle characteristics of mouse cumulus granulosa cells were determined before, during and following their expansion and mucification in vivo and in vitro. Cumulus-oocyte complexes (COC) were recovered from ovarian follicles or oviducts of prepubertal mice previously injected with pregnant mare serum gonadotrophin (PMSG) or a mixture of PMSG and human chorionic gonadotrophin (PMSG+hCG) to synchronize follicle differentiation and ovulation. Cell cycle parameters were determined by monitoring DNA content of cumulus cell nuclei, collected under rigorously controlled conditions, by flow cytometry. The proportion of cumulus cells in three cell cycle-related populations (G0/G1; S; G2/M) was calculated before and after exposure to various experimental conditions in vivo or in vitro. About 30% of cumulus cells recovered from undifferentiated (compact) COC isolated 43-45 h after PMSG injections were in S phase and 63% were in G0/G1 (2C DNA content). Less than 10% of the cells were in the G2/M population. Cell cycle profiles of cumulus cells recovered from mucified COC (oviducal) after PMSG+hCG-induced ovulation varied markedly from those collected before hCG injection and were characterized by the relative absence of S-phase cells and an increased proportion of cells in G0/G1. Cell cycle profiles of cumulus cells collected from mucified COC recovered from mouse ovarian follicles before ovulation (9-10 h after hCG) were also characterized by loss of S-phase cells and an increased G0/G1 population. Results suggest that changes in cell cycle parameters in vivo are primarily mediated in response to physiological changes that occur in the intrafollicular environment initiated by the ovulatory stimulus. A similar lack of S-phase cells was observed in mucified cumulus cells collected 24 h after exposure in vitro of compact COC to dibutyryl cyclic adenosine monophosphate (DBcAMP), follicle-stimulating hormone or epidermal growth factor (EGF). Additionally, the proportion of cumulus cells in G2/M was enhanced in COC exposed to DBcAMP, suggesting that cell division was inhibited under these conditions. Thus, both the G1-->S-phase and G2-->M-phase transitions in the cell cycle appear to be amenable to physiological regulation. Time course studies revealed dose-dependent changes in morphology occurred within 6 h of exposure in vitro of COC to EGF or DBcAMP. Results suggest that the disappearance of the S-phase population is a consequence of a decline in the number of cells beginning DNA synthesis and exit of cells from the S phase following completion of DNA synthesis. Furthermore, loss of proliferative activity in cumulus cells appears to be closely associated with COC expansion and mucification, whether induced under physiological conditions in vivo or in response to a range of hormonal stimuli in vitro. The observations indicate that several signal-transducing pathways mediate changes in cell cycle parameters during cumulus cell differentiation.

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Xeroderma Pigmentosum as a Model for Treatment of Bone Marrow Failure in Fanconi Anemia and Aplastic Anemia.

Blood ◽

10.1182/blood.v104.11.4235.4235 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4235-4235

Author(s):

W. Clark Lambert ◽

Santiago A. Centurion

Keyword(s):

Cell Cycle ◽

Bone Marrow ◽

Fanconi Anemia ◽

Xeroderma Pigmentosum ◽

Bone Marrow Failure ◽

S Phase ◽

White Female ◽

Cross Linking

Abstract We have previously shown that the primary cell cycle defect in the inherited, cancer-prone, bone marrow failure associated disease, Fanconi anemia (FA), is not in the G2 phase of the cell cycle, as had been thought for many years, but rather in the S phase. FA cells challenged with the DNA cross-linking agent, psoralen coupled with long wavelength, ultraviolet (UVA) radiation (PUVA), fail to slow their progression through the S phase of the subsequent cell cycle, as do normal cells. FA cells are extremely sensitive to the cytotoxic and clastogenic effects of DNA cross-linkers, such as PUVA, so much so that the diagnosis of FA is based on an assay, the “DEB test”, in which cells are examined for clastogenic and cytotoxic effects of diepoxybutane (DEB), a DNA cross-linking agent. More recently, we have shown that artificially slowing the cell cycle of FA cells exposed to PUVA by subsequent treatment with agents which slow their progression through S phase leads to markedly increased viability and reduced chromosome breakage in vitro. We now show that similar results can be obtained in vivo in patients with another DNA repair deficiency disease, xeroderma pigmentosum (XP), a recessively inherited disorder associated with defective repair of sunlight induced adducts in the DNA of sun-exposed tissues followed by development of numerous mutations causing large numbers of cancers in these same tissues. We treated two patients with XP, a light complected black male and a white female, both 14 years of age, in sun-exposed areas with 5-fluorouracil, an inhibitor of DNA synthesis, daily for three months. In contrast to normal patients, who only show clinical results if an inflammatory response is invoked, marked improvement in the clinical appearance of the skin was seen with no inflammation observed. This effect was confirmed histologically by examining epidermis adjacent to excised lesions in sun-exposed areas and further verified by computerized image analysis. Treatment with agents that slow progression through S phase, such as hydroxyurea, may similarly improve clinical outcomes in patients with FA or others who are developing bone marrow failure.

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Thrombin Enhances Spontaneous Tumor Growth and Cell Cycle Activation by Downregulation of p27Kip1 and Upregulation of Skp2 and MiR-222

Blood ◽

10.1182/blood.v112.11.396.396 ◽

2008 ◽

Vol 112 (11) ◽

pp. 396-396

Author(s):

Liang Hu ◽

Sherif Ibrahim ◽

Cynthia Liu ◽

Jeffrey Skaar ◽

Michelle Pagano ◽

...

Keyword(s):

Cell Cycle ◽

Growth Factors ◽

Cell Growth ◽

Tumor Growth ◽

Tumor Cell ◽

Tumor Volume ◽

S Phase ◽

Down Regulation

Abstract Although it has been generally accepted that hypercoagulability contributes to enhancing tumor growth via generation of thrombin (Cancer Cell10:355, 2006), it has not been rigorously proven, nor has the mechanism been established at the cell cycle level. Previous studies have employed thrombin-treated tumor cell lines in vitro and in vivo. In vitro studies were performed in the presence of serum which contains a panoply of growth factors. In vivo studies have used huge non-pathologic concentrations of tumor cells injected into the flank, organ or blood of a mouse. In these situations, tumor growth could be a result of thrombin-induced angiogenesis. We therefore employed a transgenic mouse prostate cancer model (TRAMP) programmed to develop prostate CA over a period of 140–175 days. We treated these animals with thrombin to induce hypercoagulability or hirudin to inhibit endogenous thrombin production, to determine whether thrombin regulates this process independent of angiogenesis. Repetitive thrombin injection enhanced prostate tumor volume 6–8 fold (p<0.04). Repetitive hirudin decreased tumor volume 13–24 fold (p<0.04) via its effect on generated endogenous thrombin, n=6. Thrombin enhanced the production of several vascular growth factors and receptors 2.5 – 3 fold in the liver (VEGF, KDR, ANG-2, Tie2, GRO-1, CD31) and enhanced angiogenesis in the liver, n=3–4. Thrombin had no effect on tumor angiogenesis. Thus, the thrombin-induced spontaneous tumor growth was independent of angiogenesis. We next turned our attention to cell cycle regulators in serum-starved (72 hr) Go-synchronized LNcap prostate CA cells, employing Brdu and Propidium iodide staining. Addition of thrombin (0.5 u/ml) or its PAR-1 receptor agonist, TFLLRN (100 uM) had the same effect as androgen containing serum, inducing cells to leave Go, enter G1 and progress to S-phase. At 8 hrs the number of S-phase cells increased dramatically for both the serum (29 fold) as well as thrombin-treated cells (48 fold), n=3. Similar observations were noted in a Glioblastoma cell line, T98G. We further analyzed the effect of thrombin by performing immunoblots on cell cycle components mediated during cell growth and proliferation. In synchronized Go cells, levels of p27Kip1, a cyclin-dependent kinase inhibitor are high, while levels of cyclins D1 and A, the activation subunits for cyclin-dependent kinases are low. Both thrombin or serum addition led to down-regulation of p27Kip1 with concomitant induction of Skp2, the E3 ubiquitin ligase for p27Kip1. Cyclins D1 and A are induced by similar kinetics, indicating entry into S-phase by 8 hrs. Since p27Kip1 appears to be a rate-limiting down-regulator of the cell cycle (absent with high tumor grade and predicts poor prognosis), we confirmed its role by testing the effect of thrombin or TFLLRN by transfecting p27Kip1 in LNcap cells. This transfection completely prevented the cell cycle stimulation induced by these agonists. A similar approach was used with Skp2 knock down (KD), a negative down-regulator of p27Kip1. KD of Skp2 (over expressed in numerous cancers) completely prevented cell cycle progression induced by thrombin/TFLLRN. MiRNA 222 (upregulated in many cancers) is another down-regulator of p27Kip1. Further analysis following thrombin treatment revealed a robust upregulation at 4 and 8 hrs, providing further proof for the role of thrombin in down-regulating p27Kip1 and stimulating tumor cell entrance into S-phase. Thus, 1) Thrombin enhances spontaneous prostate cell growth in vivo in the absence of enhanced angiogenesis; 2) Thrombin activates the tumor cell cycle by stimulating the down-regulation of p27Kip1 through the upregulation of Skp2 and MiRNA 222.

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Targeting the Cyclin D - CDK4/6 - Rb Axis in Mantle Cell Lymphoma with the Novel Translation Inhibitor Silvestrol,

Blood ◽

10.1182/blood.v118.21.3498.3498 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3498-3498

Author(s):

Lapo Alinari ◽

Ryan B. Edwards ◽

Courtney J. Prince ◽

William H. Towns ◽

Rajeswaran Mani ◽

...

Keyword(s):

Cell Cycle ◽

Cyclin D1 ◽

Tumor Cells ◽

Cell Lymphoma ◽

S Phase ◽

Cyclin D ◽

Mantle Cell ◽

Phase Entry

Abstract Abstract 3498 During cell cycle progression, D class cyclins activate cyclin dependent kinases (CDK) 4 and 6 to phosphorylate and inactivate Rb, allowing E2F-1 mediated transcription of additional cell cycle genes including cyclin E to drive S phase entry. This critical pathway is nearly universally dysregulated in cancer, providing tumor cells a strong growth advantage and escape from normal mitotic control. Substantial research is being directed toward targeting this pathway in many cancer types, with some preliminary successes being achieved with pharmacologic inhibitors of CDK4/6. However the development of alternative strategies to block this pathway could potentially provide broad therapeutic benefit. A prime example of a tumor with a disrupted cyclin D axis is Mantle Cell Lymphoma (MCL), in which the t(11;14) translocation places CCND1, the gene for cyclin D1, under the control of an immunoglobulin promoter. This results in sustained cyclin D1 expression in tumor cells and concomitant Rb inactivation, S phase entry and cell division. MCL is a relatively uncommon subset of Non-Hodgkin Lymphoma, but accounts for a disproportionate number of deaths. Treatments are limited and relapse is nearly universal; thus, new treatment strategies are essential for this disease. Silvestrol is a structurally unique, plant-derived cyclopenta[b]benzofuran with potent in vitro and in vivo anti-tumor activity in several model systems including B-cell acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL). Silvestrol inhibits the initiation step of translation by preventing assembly of eIF4A and capped mRNA into the eIF4F complex, leading to selective loss of short half-life proteins such as Mcl-1 and cyclin D1. We therefore hypothesized that silvestrol, through the depletion of cyclin D1, would demonstrate efficacy in MCL. Silvestrol showed low nanomolar IC50 values in the JeKo-1 (13 nM), Mino (17 nM) and SP-53 (43 nM) MCL cell lines at 48 hr (MTS assay; cell death confirmed by propidium iodide flow cytometry). This potency was similar in primary MCL tumor cells. Longer exposure times substantially improved the cytotoxicity of silvestrol assessed at 48 hr (approximately 50% effect achieved with a 16 hr exposure vs. 80% effect with a 24 hr exposure), suggesting that the cellular impacts of this agent increase with exposure time. Cyclins D1 and D3 were dramatically reduced in MCL cell lines with just 10 nM silvestrol at 16 hr (cyclin D2 was undetectable in these cells), with subsequent loss of Rb phosphorylation as well as cyclin E mRNA and protein, culminating in G1 cell cycle arrest. Similar to what we previously showed in CLL and ALL cells, silvestrol treatment under these conditions also caused loss of Mcl-1 protein with concurrent mitochondrial depolarization, although the exact mechanism of silvestrol-mediated cytotoxicity in these cells is still under investigation. In an aggressive xenograft mouse model of MCL, silvestrol produced a highly significant improvement in survival [median survival of vehicle vs. silvestrol treated mice (1.5 mg/kg every 48 hr) = 27 vs. 38 days; P<0.0001] without detectable toxicity. Together, these data demonstrate that the translation inhibitor silvestrol has promising in vitro and in vivo activity in MCL preclinical models. Furthermore, as the cyclin D/CDK/Rb axis is disrupted in most tumor types, this strategy may be broadly effective in other cancers as well. Disclosures: No relevant conflicts of interest to declare.

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BRCA1 Is Phosphorylated at Serine 1497 In Vivo at a Cyclin-Dependent Kinase 2 Phosphorylation Site

Molecular and Cellular Biology ◽

10.1128/mcb.19.7.4843 ◽

1999 ◽

Vol 19 (7) ◽

pp. 4843-4854 ◽

Cited By ~ 66

Author(s):

Heinz Ruffner ◽

Wei Jiang ◽

A. Grey Craig ◽

Tony Hunter ◽

Inder M. Verma

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Kinase Activity ◽

Phosphorylation Site ◽

Cyclin A ◽

Dominant Negative ◽

Protein Kinase Activity ◽

Cyclin Dependent Kinase

ABSTRACT BRCA1 is a cell cycle-regulated nuclear protein that is phosphorylated mainly on serine and to a lesser extent on threonine residues. Changes in phosphorylation occur in response to cell cycle progression and DNA damage. Specifically, BRCA1 undergoes hyperphosphorylation during late G1 and S phases of the cell cycle. Here we report that BRCA1 is phosphorylated in vivo at serine 1497 (S1497), which is part of a cyclin-dependent kinase (CDK) consensus site. S1497 can be phosphorylated in vitro by CDK2-cyclin A or E. BRCA1 coimmunoprecipitates with an endogenous serine-threonine protein kinase activity that phosphorylates S1497 in vitro. This cellular kinase activity is sensitive to transfection of a dominant negative form of CDK2 as well as the application of the CDK inhibitors p21 and butyrolactone I but not p16. Furthermore, BRCA1 coimmunoprecipitates with CDK2 and cyclin A. These results suggest that the endogenous kinase activity is composed of CDK2-cyclin complexes, at least in part, concordant with the G1/S-specific increase in BRCA1 phosphorylation.

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A CDK-regulated chromatin segregase promoting chromosome replication

Nature Communications ◽

10.1038/s41467-021-25424-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Erika Chacin ◽

Priyanka Bansal ◽

Karl-Uwe Reusswig ◽

Luis M. Diaz-Santin ◽

Pedro Ortega ◽

...

Keyword(s):

Atp Hydrolysis ◽

Genome Instability ◽

Chromosome Replication ◽

S Phase ◽

Chromatin Dynamics ◽

Replicative Stress ◽

Cdk Phosphorylation ◽

Nucleosome Disassembly

AbstractThe replication of chromosomes during S phase is critical for cellular and organismal function. Replicative stress can result in genome instability, which is a major driver of cancer. Yet how chromatin is made accessible during eukaryotic DNA synthesis is poorly understood. Here, we report the characterization of a chromatin remodeling enzyme—Yta7—entirely distinct from classical SNF2-ATPase family remodelers. Yta7 is a AAA+ -ATPase that assembles into ~1 MDa hexameric complexes capable of segregating histones from DNA. The Yta7 chromatin segregase promotes chromosome replication both in vivo and in vitro. Biochemical reconstitution experiments using purified proteins revealed that the enzymatic activity of Yta7 is regulated by S phase-forms of Cyclin-Dependent Kinase (S-CDK). S-CDK phosphorylation stimulates ATP hydrolysis by Yta7, promoting nucleosome disassembly and chromatin replication. Our results present a mechanism for how cells orchestrate chromatin dynamics in co-ordination with the cell cycle machinery to promote genome duplication during S phase.

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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

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620666200918101448 ◽

2020 ◽

Vol 20 ◽

Author(s):

Ajith J. George ◽

Bibu J. Kariyil ◽

Usha P.T. Ayyappan ◽

Anu Gopalakrishnan

Keyword(s):

Cell Cycle ◽

Cell Lines ◽

Triple Negative ◽

S Phase ◽

Chloroform Fraction ◽

Annona Muricata ◽

In Vivo Evaluation ◽

Annonaceous Acetogenins

Background: Triple negative breast cancers (TNBCs) are having high morbidity and shorter survival rate in the population. These types of cancers are having 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 anticancer effect and mechanism of action in TNBCs has not been elucidated. Objective: The present study was undertaken to evaluate the in vitro, in vivo and in silico anticancer 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 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), intra cellular reactive oxygen species assay, DNA fragmentation assay, agarose gel electrophoresis, terminal deoxynucleotidyl transferase dUTP nick end labelling (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 tumour model was also conducted. Phytoconstituents in CMAM was analysed 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±0.16 µg/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+0.04 µg/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 reduction in tumour volume and serum biomarkers. Histopathologically there was a marked reduction in cellularity, nuclear chromatin condensation and a few normal cells in 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 mitochondria mediated 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 anticanceros effect of CMAM. Thus muricatacin could be a potential candidate for the targeted therapy of TNBCs.

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