Regulation of Cell Cycle to Stimulate Adult Cardiomyocyte Proliferation and Cardiac Regeneration.

Human diseases are often caused by loss of somatic cells that are incapable of re-entering the cell cycle for regenerative repair. Here, we report a combination of cell-cycle regulators that induce stable cytokinesis in adult post-mitotic cells. We screened cell-cycle regulators expressed in proliferating fetal cardiomyocytes and found that overexpression of cyclin-dependent kinase 1 (CDK1), CDK4, cyclin B1, and cyclin D1 efficiently induced cell division in post-mitotic mouse, rat, and human cardiomyocytes. Overexpression of the cell-cycle regulators was self-limiting through proteasome-mediated degradation of the protein products. In vivo lineage tracing revealed that 15%-20% of adult cardiomyocytes expressing the four factors underwent stable cell division, with significant improvement in cardiac function after acute or subacute myocardial infarction. Chemical inhibition of Tgf-ß and Wee1 made CDK1 and cyclin B dispensable. These findings reveal a discrete combination of genes that can efficiently unlock the proliferative potential in cells that have terminally exited the cell cycle.

CDK5-cyclin B1 regulates mitotic fidelity.

CDK1 has been known to be the sole cyclin-dependent kinase (CDK) partner of cyclin B1 to drive mitotic progression1. Here we demonstrate that CDK5 is active during mitosis and is necessary for maintaining mitotic fidelity. CDK5 is an atypical CDK owing to its high expression in post-mitotic neurons and activation by non-cyclin proteins p35 and p392. Here, using independent chemical genetic approaches, we specifically abrogated CDK5 activity during mitosis, and observed mitotic defects, nuclear atypia and substantial alterations in the mitotic phosphoproteome. Notably, cyclin B1 is a mitotic co-factor of CDK5. Computational modelling, comparison with experimentally derived structures of CDK-cyclin complexes and validation with mutational analysis indicate that CDK5-cyclin B1 can form a functional complex. Disruption of the CDK5-cyclin B1 complex phenocopies CDK5 abrogation in mitosis. Together, our results demonstrate that cyclin B1 partners with both CDK5 and CDK1, and CDK5-cyclin B1 functions as a canonical CDK-cyclin complex to ensure mitotic fidelity.

Spatial control of the APC/C ensures the rapid degradation of cyclin B1.

The proper control of mitosis depends on the ubiquitin-mediated degradation of the right mitotic regulator at the right time. This is effected by the Anaphase Promoting Complex/Cyclosome (APC/C) ubiquitin ligase that is regulated by the Spindle Assembly Checkpoint (SAC). The SAC prevents the APC/C from recognising Cyclin B1, the essential anaphase and cytokinesis inhibitor, until all chromosomes are attached to the spindle. Once chromosomes are attached, Cyclin B1 is rapidly degraded to enable chromosome segregation and cytokinesis. We have a good understanding of how the SAC inhibits the APC/C, but relatively little is known about how the APC/C recognises Cyclin B1 as soon as the SAC is turned off. Here, by combining live-cell imaging, in vitro reconstitution biochemistry, and structural analysis by cryo-electron microscopy, we provide evidence that the rapid recognition of Cyclin B1 in metaphase requires spatial regulation of the APC/C. Using fluorescence cross-correlation spectroscopy, we find that Cyclin B1 and the APC/C primarily interact at the mitotic apparatus. We show that this is because Cyclin B1, like the APC/C, binds to nucleosomes, and identify an 'arginine-anchor' in the N-terminus as necessary and sufficient for binding to the nucleosome. Mutating the arginine anchor on Cyclin B1 reduces its interaction with the APC/C and delays its degradation: cells with the mutant, non-nucleosome-binding Cyclin B1 become aneuploid, demonstrating the physiological relevance of our findings. Together, our data demonstrate that mitotic chromosomes promote the efficient interaction between Cyclin B1 and the APC/C to ensure the timely degradation of Cyclin B1 and genomic stability.

Multiple intersecting pathways are involved in CPEB1 phosphorylation and regulation of translation during mouse oocyte meiosis.

The RNA-binding protein cytoplasmic polyadenylation element binding 1 (CPEB1) plays a fundamental role in regulating mRNA translation in oocytes. However, the specifics of how and which protein kinase cascades modulate CPEB1 activity are still controversial. Using genetic and pharmacological tools, and detailed time courses, we have re-evaluated the relationship between CPEB1 phosphorylation and translation activation during mouse oocyte maturation. We show that both the CDK1/MAPK and AURKA/PLK1 pathways converge on CPEB1 phosphorylation during prometaphase of meiosis I. Only inactivation of the CDK1/MAPK pathway disrupts translation, whereas inactivation of either pathway alone leads to CPEB1 stabilization. However, CPEB1 stabilization induced by inactivation of the AURKA/PLK1 pathway does not affect translation, indicating that destabilization and/or degradation is not linked to translational activation. The accumulation of endogenous CCNB1 protein closely recapitulates the translation data that use an exogenous template. These findings support the overarching hypothesis that the activation of translation during prometaphase in mouse oocytes relies on a CDK1/MAPK-dependent CPEB1 phosphorylation, and that translational activation precedes CPEB1 destabilization.

Spatial positive feedback at the onset of mitosis.

Mitosis is triggered by the activation of Cdk1-cyclin B1 and its translocation from the cytoplasm to the nucleus. Positive feedback loops regulate the activation of Cdk1-cyclin B1 and help make the process irreversible and all-or-none in character. Here we examine whether an analogous process, spatial positive feedback, regulates Cdk1-cyclin B1 redistribution. We used chemical biology approaches and live-cell microscopy to show that nuclear Cdk1-cyclin B1 promotes the translocation of Cdk1-cyclin B1 to the nucleus. Mechanistic studies suggest that cyclin B1 phosphorylation promotes nuclear translocation and, conversely, nuclear translocation promotes cyclin B1 phosphorylation, accounting for the feedback. Interfering with the abruptness of Cdk1-cyclin B1 translocation affects the timing and synchronicity of subsequent mitotic events, underscoring the functional importance of this feedback. We propose that spatial positive feedback ensures a rapid, complete, robust, and irreversible transition from interphase to mitosis and suggest that bistable spatiotemporal switches may be widespread in biological regulation.

Structural basis of human separase regulation by securin and CDK1-cyclin B1.

In early mitosis, the duplicated chromosomes are held together by the ring-shaped cohesin complex1. Separation of chromosomes during anaphase is triggered by separase-a large cysteine endopeptidase that cleaves the cohesin subunit SCC1 (also known as RAD212-4). Separase is activated by degradation of its inhibitors, securin5 and cyclin B6, but the molecular mechanisms of separase regulation are not clear. Here we used cryogenic electron microscopy to determine the structures of human separase in complex with either securin or CDK1-cyclin B1-CKS1. In both complexes, separase is inhibited by pseudosubstrate motifs that block substrate binding at the catalytic site and at nearby docking sites. As in Caenorhabditis elegans7 and yeast8, human securin contains its own pseudosubstrate motifs. By contrast, CDK1-cyclin B1 inhibits separase by deploying pseudosubstrate motifs from intrinsically disordered loops in separase itself. One autoinhibitory loop is oriented by CDK1-cyclin B1 to block the catalytic sites of both separase and CDK19,10. Another autoinhibitory loop blocks substrate docking in a cleft adjacent to the separase catalytic site. A third separase loop contains a phosphoserine6 that promotes complex assembly by binding to a conserved phosphate-binding pocket in cyclin B1. Our study reveals the diverse array of mechanisms by which securin and CDK1-cyclin B1 bind and inhibit separase, providing the molecular basis for the robust control of chromosome segregation.

Mitotic CDK Promotes Replisome Disassembly, Fork Breakage, and Complex DNA Rearrangements.

DNA replication errors generate complex chromosomal rearrangements and thereby contribute to tumorigenesis and other human diseases. One mechanism that triggers these errors is mitotic entry before the completion of DNA replication. To address how mitosis might affect DNA replication, we used Xenopus egg extracts. When mitotic CDK (Cyclin B1-CDK1) is used to drive interphase egg extracts into a mitotic state, the replicative CMG (CDC45/MCM2-7/GINS) helicase undergoes ubiquitylation on its MCM7 subunit, dependent on the E3 ubiquitin ligase TRAIP. Whether replisomes have stalled or undergone termination, CMG ubiquitylation is followed by its extraction from chromatin by the CDC48/p97 ATPase. TRAIP-dependent CMG unloading during mitosis is also seen in C. elegans early embryos. At stalled forks, CMG removal results in fork breakage and end joining events involving deletions and templated insertions. Our results identify a mitotic pathway of global replisome disassembly that can trigger replication fork collapse and DNA rearrangements.

Alternative cleavage and polyadenylation of the Ccnb1 mRNA defines accumulation of cyclin protein during the meiotic cell cycle.

Progression through the mitotic and meiotic cell cycle is driven by fluctuations in the levels of cyclins, the regulatory subunits controlling the localization and activity of CDK1 kinases. Cyclin levels are regulated through a precise balance of synthesis and degradation. Here we demonstrate that the synthesis of Cyclin B1 during the oocyte meiotic cell cycle is defined by the selective translation of mRNA variants generated through alternative cleavage and polyadenylation (APA). Using gene editing in mice, we introduced mutations into the proximal and distal polyadenylation elements of the 3' untranslated region (UTR) of the Ccnb1 mRNA. Through in vivo loss-of-function experiments, we demonstrate that the translation of mRNA with a short 3' UTR specifies Cyclin B1 protein levels that set the timing of meiotic re-entry. In contrast, translation directed by a long 3' UTR is necessary to direct Cyclin B1 protein accumulation during the MI/MII transition. These findings establish that the progression through the cell cycle is dependent on the selective translation of multiple mRNA variants generated by APA.

Kielin/chordin-like protein deficiency aggravates pressure overload-induced cardiac dysfunction and remodeling via P53/P21/CCNB1 signaling in mice.

Targeting cardiac remodeling is regarded as a key therapeutic strategy for heart failure. Kielin/chordin-like protein (KCP) is a secretory protein with 18 cysteine-rich domains and associated with kidney and liver fibrosis. However, the relationship between KCP and cardiac remodeling remains unclear. Here, we aimed to investigate the role of KCP in cardiac remodeling induced by pressure overload and explore its potential mechanisms. Left ventricular (LV) KCP expression was measured with real-time quantitative PCR, western blotting, and immunofluorescence staining in pressure overload-induced cardiac remodeling in mice. Cardiac function and remodeling were evaluated in wide-type (WT) mice and KCP knockout (KO) mice by echocardiography, which were further confirmed by histological analysis with hematoxylin and eosin and Masson staining. RNA sequence was performed with LV tissue from WT and KO mice to identify differentially expressed genes and related signaling pathways. Primary cardiac fibroblasts (CFs) were used to validate the regulatory role and potential mechanisms of KCP during fibrosis. KCP was down-regulated in the progression of cardiac remodeling induced by pressure overload, and was mainly expressed in fibroblasts. KCP deficiency significantly aggravated pressure overload-induced cardiac dysfunction and remodeling. RNA sequence revealed that the role of KCP deficiency in cardiac remodeling was associated with cell division, cell cycle, and P53 signaling pathway, while cyclin B1 (CCNB1) was the most significantly up-regulated gene. Further investigation in vivo and in vitro suggested that KCP deficiency promoted the proliferation of CFs via P53/P21/CCNB1 pathway. Taken together, these results suggested that KCP deficiency aggravates cardiac dysfunction and remodeling induced by pressure overload via P53/P21/CCNB1 signaling in mice.

BRCA1 deficiency enhances the aggressiveness of breast cancer cells expressing HPV16 oncoproteins.

BACKGROUND INFORMATION: The precise etiology of breast cancer is not completely understood, although women with BRCA1 gene mutations have a significantly increased risk of developing the disease. In addition, sporadic breast cancer is frequently associated with decreased BRCA1 gene expression. Growing evidence of Human papillomaviruses (HPVs) infections in breast tumors has raised the possibility of the involvement of HPVs in the pathogenesis of breast cancer. We investigated whether the effects of HPV oncoproteins E6 and E7 were influenced by the expression levels of BRCA1. HPV16E6E7 (prototype or E6D25E/E7N29S Asian variant type) were stably expressed in MDA-MB231 breast cancer cells, wild type for BRCA1, or with BRCA1 knocked down. RESULTS: Expression of HPV16E6E7 oncogenes did not affect BRCA1 levels and the abundance of HPV16E6E7 was not altered by BRCA1 knockdown. BRCA1 levels did not alter HPV16E6E7-dependent degradation of G1-S cell cycle proteins p53 and pRb. However, we found that the expression of G2-M cell cycle protein cyclin B1 enhanced by HPV16E6E7 was impacted by BRCA1 levels. Especially, we found the correlation between BRCA1 and cyclin B1 expression and this was also confirmed in breast cancer samples from a Thai cohort. We further demonstrated that the combination of HPV oncoproteins and low levels of BRCA1 protein appears to enhance proliferation and invasion. Transactivation activities of HPV16E6E7 on genes regulating cell proliferation and invasion (TGF-ß and vimentin) were significantly increased in BRCA1-deficient cells. CONCLUSIONS: Our results indicate that a deficiency of BRCA1 promotes the transactivation activity of HPV16E6E7 leading to increase of cell proliferation and invasion. SIGNIFICANCE: HPV infection appears to have the potential to enhance the aggressiveness of breast cancers, especially those deficient in BRCA1.

Inhibition of vinculin activity has an adverse effect on porcine ovarian cells.

The existing knowledge of the involvement of vinculin (VCL) in the control of ovarian cell functions is insufficient. To understand the role of VCL in the control of basic porcine ovarian granulosa cell functions, we decreased VCL activity by small interfering RNA (VCL siRNA). The expression of VCL, accumulation of VCL protein, cell viability, proliferation (accumulation of PCNA and cyclin B1), proportion of proliferative active cells, apoptosis (accumulation of bax, caspase 3, p53, antiapoptotic marker bcl2, and bax/bcl-2 ratio), DNA fragmentation, and release of steroid hormones and IGF-I were analyzed by RT‒qPCR, Trypan blue exclusion test, quantitative immunocytochemistry, XTT assay, TUNEL assay, and ELISA. The suppression of VCL activity inhibited cell viability, the accumulation of the proliferation-related proteins PCNA and cyclin B1, the antiapoptotic protein bcl2, and the proportion of proliferative active cells. Moreover, VCL siRNA inhibited the release of progesterone, estradiol, and IGF-1. VCL siRNA increased the proportion of the proapoptotic proteins bax, caspase 3, p53, the proportion of DNA fragmented cells, and stimulated testosterone release. Taken together, the present study is the first evidence that inhibition of VCL suppresses porcine granulosa cell functions. Moreover, the results suggest that VCL can be a potent physiological stimulator of ovarian functions.

Maternal mRNAs with distinct 3' UTRs define the temporal pattern of Ccnb1 synthesis during mouse oocyte meiotic maturation.

The final stages of female gamete maturation occur in the virtual absence of transcription, with gene expression driven by a program of selective unmasking, translation, and degradation of maternal mRNAs. Here we demonstrate that the timing of Ccnb1 mRNA translation in mouse oocytes is dependent on the presence of transcripts with different 3' untranslated regions (UTRs). This 3' UTR heterogeneity directs distinct temporal patterns of translational activation or repression. Inclusion or exclusion of cis-acting elements is responsible for these divergent regulations. Our findings reveal an additional layer of translation control through alternative polyadenylation usage required to fine-tune the timing of meiosis progression.

Targeting PNPO to suppress tumor growth via inhibiting autophagic flux and to reverse paclitaxel resistance in ovarian cancer.

Our previous study showed that pyridoxine 5'-phosphate oxidase (PNPO) is a tissue biomarker of ovarian cancer (OC) and has a prognostic implication but detailed mechanisms remain unclear. The current study focused on PNPO-regulated lysosome/autophagy-mediated cellular processes and the potential role of PNPO in chemoresistance. We found that PNPO was overexpressed in OC cells and was a prognostic factor in OC patients. PNPO significantly promoted cell proliferation via the regulation of cyclin B1 and phosphorylated CDK1 and shortened the G2M phase in a cell cycle. Overexpressed PNPO enhanced the biogenesis and perinuclear distribution of lysosomes, promoting the degradation of autophagosomes and boosting the autophagic flux. Further, an autolysosome marker LAMP2 was upregulated in OC cells. Silencing LAMP2 suppressed cell growth and induced cell apoptosis. LAMP2-siRNA blocked PNPO action in OC cells, indicating that the function of PNPO on cellular processes was mediated by LAMP2. These data suggest the existence of the PNPO-LAMP2 axis. Moreover, silencing PNPO suppressed xenographic tumor formation. Chloroquine counteracted the promotion effect of PNPO on autophagic flux and inhibited OC cell survival, facilitating the inhibitory effect of PNPO-shRNA on tumor growth in vivo. Finally, PNPO was overexpressed in paclitaxel-resistant OC cells. PNPO-siRNA enhanced paclitaxel sensitivity in vitro and in vivo. In conclusion, PNPO has a regulatory effect on lysosomal biogenesis that in turn promotes autophagic flux, leading to OC cell proliferation, and tumor formation, and is a paclitaxel-resistant factor. These data imply a potential application by targeting PNPO to suppress tumor growth and reverse PTX resistance in OC.

Spindle checkpoint: trapped by the corona, cyclin B1 goes MAD.

The spindle checkpoint protects against aneuploidy by ensuring that dividing cells only proceed with chromosome segregation once all kinetochores are stably attached to spindle microtubules. The checkpoint protein MAD1 localizes to the corona, a structural expansion of the kinetochore forming in the absence of microtubule attachment, but molecular mechanism or functional significance of this localization remains unknown. Recent results now show that cyclin B1 recruits MAD1 to the corona and that this MAD1 pool is required for robust checkpoint signaling.

Cyclin B1 scaffolds MAD1 at the kinetochore corona to activate the mitotic checkpoint.

Cyclin B:CDK1 is the master kinase regulator of mitosis. We show here that, in addition to its kinase functions, mammalian Cyclin B also scaffolds a localised signalling pathway to help preserve genome stability. Cyclin B1 localises to an expanded region of the outer kinetochore, known as the corona, where it scaffolds the spindle assembly checkpoint (SAC) machinery by binding directly to MAD1. In vitro reconstitutions map the key binding interface to a few acidic residues in the N-terminal region of MAD1, and point mutations in this sequence abolish MAD1 corona localisation and weaken the SAC. Therefore, Cyclin B1 is the long-sought-after scaffold that links MAD1 to the corona, and this specific pool of MAD1 is needed to generate a robust SAC response. Robustness arises because Cyclin B1:MAD1 localisation loses dependence on MPS1 kinase after the corona has been established, ensuring that corona-localised MAD1 can still be phosphorylated when MPS1 activity is low. Therefore, this study explains how corona-MAD1 generates a robust SAC signal, and it reveals a scaffolding role for the key mitotic kinase, Cyclin B1:CDK1, which ultimately helps to inhibit its own degradation.

Phosphorylation of VP1 Mediated by CDK1-Cyclin B1 Facilitates Infectious Bursal Disease Virus Replication.

Infectious bursal disease virus (IBDV) is a double-stranded RNA (dsRNA) virus belonging to the genus Avibirnavirus in the family Birnaviridae. It can cause serious failure of vaccination in young poultry birds with impaired immune systems. Post-translational modifications of the VP1 protein are essential for viral RNA transcription, genome replication, and viral multiplication. Little information is available so far regarding the exact mechanism of phosphorylation of IBDV VP1 and its significance in the viral life cycle. Here, we provide several lines of evidence that the cyclin-dependent kinase 1 (CDK1)-cyclin B1 complex phosphorylates VP1, which facilitates viral replication. We show that the CDK1-cyclin B1 specifically interacts with VP1 and phosphorylates VP1 on the serine 7 residue, located in the N-terminal 7SPAQ10 region, which follows the optimal phosphorylation motif of CDK1, p-S/T-P. Additionally, IBDV infection drives the cytoplasmic accumulation of CDK1-cyclin B1, which co-localizes with VP1, supporting the kinase activity of CDK1-cyclin B1. Treatment with CDK1 inhibitor RO3306 and knockdown of CDK1-cyclin B1 severely disrupts the polymerase activity of VP1, resulting in diminished viral replication. Moreover, the replication of S7A mutant recombinant IBDV was significantly decreased compared to that of wild-type (WT) IBDV. Thus, CDK1-cyclin B1 is a crucial enzyme which phosphorylates IBDV VP1 on serine 7, which is necessary both for the polymerase activity of VP1 and for viral replication. IMPORTANCE Infectious bursal disease virus still poses a great economic threat to the global poultry farming industry. Detailed information on the steps of viral genome replication is essential for the development of antiviral therapeutics. Phosphorylation is a common post-translational modification in several viral proteins. There is a lack of information regarding the significance of VP1 phosphorylation and its role in modulating the viral life cycle. In this study, we found that CDK1-cyclin B1 accumulates in the cytoplasm and phosphorylates VP1 on serine 7. The presence of a CDK1 inhibitor and the silencing of CDK1-cyclin B1 decrease IBDV replication. The mutation of VP1 serine 7 to alanine reduces VP1 polymerase activity, disrupting the viral life cycle, which suggests that this residue serves an essential function. Our study offers novel insights into the regulatory mechanism of VP1 phosphorylation.

MEIKIN expression and its C-terminal phosphorylation by PLK1 is closely related the metaphase-anaphase transition by affecting cyclin B1 and Securin stabilization in meiotic oocyte.

Oocyte meiotic maturation failure and chromosome abnormality is one of the main causes of infertility, abortion, and diseases. The mono-orientation of sister chromatids during the first meiosis is important for ensuring accurate chromosome segregation in oocytes. MEIKIN is a germ cell-specific protein that can regulate the mono-orientation of sister chromatids and the protection of the centromeric cohesin complex during meiosis I. Here we found that MEIKIN is a maternal protein that was highly expressed in mouse oocytes before the metaphase I (MI) stage, but became degraded by the MII stage and dramatically reduced after fertilization. Strikingly, MEIKIN underwent phosphorylation modification after germinal vesicle breakdown (GVBD), indicating its possible function in subsequent cellular event regulation. We further showed that MEIKIN phosphorylation was mediated by PLK1 at its carboxyl terminal region and its C-terminus was its key functional domain. To clarify the biological significance of meikin degradation during later stages of oocyte maturation, exogenous expression of MEIKIN was employed, which showed that suppression of MEIKIN degradation resulted in chromosome misalignment, cyclin B1 and Securin degradation failure, and MI arrest through a spindle assembly checkpoint (SAC)-independent mechanism. Exogenous expression of MEIKIN also inhibited metaphase II (MII) exit and early embryo development. These results indicate that proper MEIKIN expression level and its C-terminal phosphorylation by PLK1 are critical for regulating the metaphase-anaphase transition in meiotic oocyte. The findings of this study are important for understanding the regulation of chromosome segregation and the prevention meiotic abnormality.

B1-type cyclins control microtubule organization during cell division in Arabidopsis.

Flowering plants contain a large number of cyclin families, each containing multiple members, most of which have not been characterized to date. Here, we analyzed the role of the B1 subclass of mitotic cyclins in cell cycle control during Arabidopsis development. While we reveal CYCB1;5 to be a pseudogene, the remaining four members were found to be expressed in dividing cells. Mutant analyses showed a complex pattern of overlapping, development-specific requirements of B1-type cyclins with CYCB1;2 playing a central role. The double mutant cycb1;1 cycb1;2 is severely compromised in growth, yet viable beyond the seedling stage, hence representing a unique opportunity to study the function of B1-type cyclin activity at the organismic level. Immunolocalization of microtubules in cycb1;1 cycb1;2 and treating mutants with the microtubule drug oryzalin revealed a key role of B1-type cyclins in orchestrating mitotic microtubule networks. Subsequently, we identified the GAMMA-TUBULIN COMPLEX PROTEIN 3-INTERACTING PROTEIN 1 (GIP1/MOZART) as an in vitro substrate of B1-type cyclin complexes and further genetic analyses support a potential role in the regulation of GIP1 by CYCB1s.

Serum/glucocorticoid-regulated kinase 1 contributes to the proliferation of varicella-zoster virus and induction of cyclin B1 expression.

In this study, we investigated the role of serum/glucocorticoid-regulated kinase 1 (SGK1) in varicella-zoster virus (VZV) replication. VZV DNA replication and plaque formation were inhibited by SGK1 knockout and treatment with an SGK1 inhibitor. Furthermore, SGK1 inhibition suppressed the increase in cyclin B1 expression induced by VZV infection. These results suggest that VZV infection induces SGK1 activation, which is required for efficient viral proliferation through the expression of cyclin B1. This is the first study to report that SGK1 is involved in the VZV life cycle.

The effects of enoxaparin treatment in a xenograft mouse model of oral squamous cell carcinoma: A pilot study.

BACKGROUND: Recent studies suggest that enoxaparin may have therapeutic effects on oral squamous cell carcinoma. We aimed to assess this effect utilizing xenograft mouse model through evaluations of proliferation and angiogenesis markers at the RNA and protein levels. METHODS: Mice were divided into enoxaparin treatment (n = 4), positive control (n = 4) and negative control (n = 3) groups. Immunohistochemical analyses were performed utilizing Bcl-2, Bax and Ki-67 antibodies. Expression levels of proliferation and apoptosis related genes were calculated utilizing qRT-PCR. Time-dependent proliferation assays were performed in OSC-19 and HEK293 cell-lines. RESULTS: Bax antibody showed positive staining in the cytoplasm and nuclei of tumor cells, while Bcl-2 antibody displayed staining only in the cytoplasm. A proliferation index of 15%-20% was found in all groups with the Ki-67 marker indicating no metastasis. Enoxaparin treatment caused decrease in BCL2, BAX and CCNB1 genes' expressions. Compared to HEK293, proliferation assays demonstrated higher division rates in OSC-19 with a significant decrease in viability after 96 h. CONCLUSION: Reduced BCL-2 expression indicates a regression of tumor growth, but reduced BAX expression is not correlated with increased apoptosis. Despite the aggressive nature of OSC-19, our results showed a low cell viability with a high division rate when compared with the control HEK293. This paralleled our in vivo findings that showed absence of lymph node metastasis across all mice groups. This discrepancy with the literature suggests that further investigations of the underlying mechanisms and protein-level analyses are needed to draw definitive conclusions about the effect of enoxaparin on OSC-19 behavior.