Discovery and resistance mechanism of a selective CDK12 degrader.

Cyclin-dependent kinase 12 (CDK12) is an emerging therapeutic target due to its role in regulating transcription of DNA-damage response (DDR) genes. However, development of selective small molecules targeting CDK12 has been challenging due to the high degree of homology between kinase domains of CDK12 and other transcriptional CDKs, most notably CDK13. In the present study, we report the rational design and characterization of a CDK12-specific degrader, BSJ-4-116. BSJ-4-116 selectively degraded CDK12 as assessed through quantitative proteomics. Selective degradation of CDK12 resulted in premature cleavage and poly(adenylation) of DDR genes. Moreover, BSJ-4-116 exhibited potent antiproliferative effects, alone and in combination with the poly(ADP-ribose) polymerase inhibitor olaparib, as well as when used as a single agent against cell lines resistant to covalent CDK12 inhibitors. Two point mutations in CDK12 were identified that confer resistance to BSJ-4-116, demonstrating a potential mechanism that tumor cells can use to evade bivalent degrader molecules.

Immunotherapy for Prostate Cancer: Treatments for the "Lethal" Phenotype.

Multiple immunologic platforms have provided minimal impact in patients with metastatic castration-resistant prostate cancer, necessitating that novel approaches continue to be developed. Although checkpoint inhibitors have been largely ineffective, there remain small cohorts of patients who have durable responses but lack the conventional indicators for response to this class of drugs, that is, high mutational burden or significant genomic alterations, as seen in other solid tumors. This article presents an update on the evolution of immunotherapeutics that target a more lethal form of prostate cancer and provides the groundwork for future considerations as to how this field should proceed.

Inhibition of CD73 using folate targeted nanoparticles carrying anti-CD73 siRNA potentiates anticancer efficacy of Dinaciclib.

Conventional therapeutic methods against cancer, including chemotherapy, radiotherapy, surgery, and combination therapy, have exhibited different toxicity levels due to their unspecific mechanism of action. To overcome the challenges facing conventional cancer therapies, newly developed methods are being investigated. Significant levels of specificity, remarkable accumulation at the tumor site, limited side effects, and minimal off-target effects enable the newly synthesized nanoparticles (NPs) to become the preferred drug delivery method in anticancer therapeutic approaches. According to the literature, CD73 has a pivotal role in cancer progression and resistance to chemotherapy and radiotherapy. Therefore, CD73 has attracted considerable attention among scientists to target this molecule. Accordingly, FDA approved CDK inhibitors such as Dinaciclib that blocks CDK1, 2, 5, and 9, and exhibits significant anticancer activity. So in this study, we intended to simultaneously suppress CD73 and CDKs in cancer cells by using the folic acid (FA)-conjugated chitosan-lactate (CL) NPs loaded with anti-CD73 siRNA and Dinaciclib to control tumor progression and metastasis. The results showed that NPs could effectively transfect cancer cells in a FA receptor-dependent manner leading to suppression of proliferation, survival, migration, and metastatic potential. Moreover, the treatment of tumor-bearing mice with this combination strategy robustly inhibited tumor growth and enhanced survival time in mice. These findings imply the high potential of FA-CL NPs loaded with anti-CD73 siRNA and Dinaciclib for use in cancer treatment shortly.

Bufalin suppresses hepatocarcinogenesis by targeting ß-catenin/TCF signaling via cell cycle-related kinase.

Hepatocellular carcinoma (HCC) is one of the most aggressive malignant tumors, of which treatment options are limited especially in advanced stage. Bufalin, the major digoxin-like component of the traditional Chinese medicine Chansu, exhibits significant antitumor activities in hepatoma cells, but the potential mechanism is obscure. Cell cycle-related kinase (CCRK) is recently identified to be a crucial oncogenic master regulator to drive hepatocarcinogenesis. Here we investigated the molecular function of bufalin on CCRK-regulated signaling pathway, and expounded the underlying mechanism in HCC suppression. In vitro with PLC5 HCC cells and human immortal LO2 cells, proliferation, malignant transformation and cell cycle progression assays were performed to evaluate the antitumor effect of bufalin. In vivo with xenograft and orthotopic mice models, tumor growths with weight and volume change were assessed with or without bufalin treatment. Western blot, RT-qPCR, immunofluorescence and immunohistochemistry were conducted to examine the expression level of CCRK and ß-catenin/TCF signaling cascade. We revealed that bufalin suppresses PLC5 HCC cell proliferation, transformation and cell cycle progression rather than LO2 cells, which is correlated with CCRK-mediated ß-catenin/TCF signaling. It was also confirmed in mice model. Thus, bufalin is a potential anti-HCC therapeutic candidate through the inhibition of CCRK-driven ß-catenin/TCF oncogenic signaling pathway.

EWS/FLI Confers Tumor Cell Synthetic Lethality to CDK12 Inhibition in Ewing Sarcoma.

Many cancer types are driven by oncogenic transcription factors that have been difficult to drug. Transcriptional inhibitors, however, may offer inroads into targeting these cancers. Through chemical genomics screening, we identified that Ewing sarcoma is a disease with preferential sensitivity to THZ1, a covalent small-molecule CDK7/12/13 inhibitor. The selective CDK12/13 inhibitor, THZ531, impairs DNA damage repair in an EWS/FLI-dependent manner, supporting a synthetic lethal relationship between response to THZ1/THZ531 and EWS/FLI expression. The combination of these molecules with PARP inhibitors showed striking synergy in cell viability and DNA damage assays in vitro and in multiple models of Ewing sarcoma, including a PDX, in vivo without hematopoietic toxicity.

Investigation of the cytotoxic effect of flavopiridol in canine lymphoma cell lines.

The cyclin-dependent kinase (CDK) inhibitor, flavopiridol, was tested as a potential new cancer therapeutic agent to treat canine lymphoma by examining its effect on cell growth of canine lymphoma cell lines in vitro. Flavopiridol induced profound cell death in all eight lymphoma cell lines at 400 nM, and in all cases cell death was due to apoptosis. Apoptosis was inhibited by caspase inhibitor, despite the variable sensitivities between cell lines. Analysis of the mechanism of flavopiridol-induced apoptosis showed that Rb phosphorylation was inhibited, possibly due to CDK4 or CDK6 inhibition. There was also decreased expression of Rb protein and anti-apoptotic proteins, Mcl-1 and XIAP, possibly through transcriptional regulation by inhibition of CDK7 or CDK9 activation. Canine lymphoma cell line-xenotransplanted mice were then treated with flavopiridol and profound tumour shrinkage was observed. This study describes a new therapeutic approach using flavopiridol for canine lymphoma treatment.

The effect of statins on cancer cells--review.

Statins [3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, abbreviated HMGCR) inhibitors], are well-known cholesterol-depleting agents. Since the early 1990 s, it has been known that statins could be successfully used in cancer therapy, but the exact mechanism(s) of statin activity remains unclear and is now an extensive focus of investigation. So far, it was proven that there are several mechanisms that are activated by statins in cancer cells; some of them are leading to cell death. Statins exert different effects depending on cell line, statin concentration, duration of exposure of cells to statins, and the type of statin being used. It was shown that statins may inhibit the cell cycle by influence on both expression and activity of proteins involved in cell-cycle progression such as cyclins, cyclin-dependent kinases (CDK), and/or inhibitors of CDK. Also, statins may induce apoptosis by both intrinsic and extrinsic pathways. Statin treatment may lead to changes in molecular pathways dependent on the EGF receptor, mainly via inhibition of isoprenoid synthesis. By inhibition of the synthesis of cholesterol, statins may destabilize the cell membrane. Moreover, statins may change the arrangement of transporter OATP1, the localization of HMGCR, and could induce conformational changes in GLUT proteins. In this review, we have tried to gather and compare most of the recent outcomes of the research in this field. We have also attempted to explain why hydrophilic statins are less effective than hydrophobic statins. Finally, we have gathered results from in vivo experiments, presenting the use of statins in combined therapies and discussed a number of molecular targets that could serve as biomarkers predisposing to statin therapy.

Diallyl disulfide induces G2/M arrest and promotes apoptosis through the p53/p21 and MEK-ERK pathways in human esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is an aggressive tumor with high incidence and mortality worldwide. Diallyl disulfide (DADS) is a natural organosulfur compound, isolated from garlic. In this study, MTT assay showed that DADS significantly reduced cell viability in a dose- and time-dependent manner in ESCC cells, with lower toxicity in normal liver cells. Cell cycle analysis revealed that DADS made G2/M phase arrest. Molecular analysis suggested that this cell cycle arrest was likely made by the decrease of cyclin B1, cdc2, p-cdc2, cdc25c in concomitance with activation of the p53/p21 pathway. Apoptosis was detected by Annexin V/PI staining. The molecule markers showed that DADS induced apoptosis through activating caspases, altering the Bax/Bcl-2 balance and suppressing the MEK-ERK pathway. Our data indicated that DADS has the potential to be an effective and safe anticancer agent for ESCC therapy in the near future.

Effects and mechanisms of total Panax notoginseng saponins on proliferation of vascular smooth muscle cells with plasma pharmacology method.

OBJECTIVES: Total Panax notoginseng saponin (TPNS) is extracted from Panax notoginseng. Our previous studies suggested that TPNS could inhibit intimal hyperplasia. This study discussed the impact of TPNS on the proliferation of vascular smooth muscle cells (VSMCs) and revealed the associated mechanisms through cell cycle-related factors and extracellular regulated protein kinase (ERK) signal transduction pathway. METHODS: A VSMC proliferation model induced by platelet-derived growth factor (PDGF) was established to observe the effects of rat drug-containing plasma on VSMC proliferation. KEY FINDINGS: After being stimulated by PDGF, the proliferating cell nuclear antigen (PCNA) and c-fos content increased, while up-regulation of cyclinD1, cyclin-dependent kinase-4 (CDK4) and down-regulation of p21 protein were observed. These changes were inhibited by atorvastatin and TSPN drug-containing plasma, and the inhibitive activity in both groups was not significant. Furthermore, both atorvastatin and TSPN could obviously inhibit the activation of PDGF-induced P-ERK1/2 and increase the content of MKP-1, there were also no significant differences. CONCLUSIONS: These results suggested that atorvastatin and TPNS could inhibit VSMC proliferation by inhibiting the activation of ERK signalling pathway.

Enhanced expression of cyclins and cyclin-dependent kinases in aniline-induced cell proliferation in rat spleen.

Aniline exposure is associated with toxicity to the spleen leading to splenomegaly, hyperplasia, fibrosis and a variety of sarcomas of the spleen on chronic exposure. In earlier studies, we have shown that aniline exposure leads to iron overload, oxidative stress and activation of redox-sensitive transcription factors, which could regulate various genes leading to a tumorigenic response in the spleen. However, molecular mechanisms leading to aniline-induced cellular proliferation in the spleen remain largely unknown. This study was, therefore, undertaken on the regulation of G1 phase cell cycle proteins (cyclins), expression of cyclin-dependent kinases (CDKs), phosphorylation of retinoblastoma protein (pRB) and cell proliferation in the spleen, in an experimental condition preceding a tumorigenic response. Male SD rats were treated with aniline (0.5 mmol/kg/day via drinking water) for 30 days (controls received drinking water only), and splenocyte proliferation, protein expression of G1 phase cyclins, CDKs and pRB were measured. Aniline treatment resulted in significant increases in splenocyte proliferation, based on cell counts, cell proliferation markers including proliferating cell nuclear antigen (PCNA), nuclear Ki67 protein (Ki67) and minichromosome maintenance (MCM), MTT assay and flow cytometric analysis. Western blot analysis of splenocyte proteins from aniline-treated rats showed significantly increased expression of cyclins D1, D2, D3 and E, as compared to the controls. Similarly, real-time PCR analysis showed significantly increased mRNA expression for cyclins D1, D2, D3 and E in the spleens of aniline-treated rats. The overexpression of these cyclins was associated with increases in the expression of CDK4, CDK6, CDK2 as well as phosphorylation of pRB protein. Our data suggest that increased expression of cyclins, CDKs and phosphorylation of pRB protein could be critical in cell proliferation, and may contribute to aniline-induced tumorigenic response in the spleen.

Novel combretastatin A-4 derivative XN0502 induces cell cycle arrest and apoptosis in A549 cells.

Combretastatin A-4 (CA-4) is a tubulin-binding compound currently in phase II trial as a tumor vascular-targeting agent. The present study evaluates the anti-tumor activities and establishes the mechanism of the action of 4-(4-methoxyphenyl)-5-(3,4,5-trimethoxyphenyl)-1H-pyrazol-3-amine(XN0502), a novel synthesized CA-4 analogue, in an effort towards finding the favorable therapeutics of CA-4 derivatives. XN0502 is characterized by its more potent anti-proliferative activities against non-small cell lung cancer A549 cells (IC(50): 1.8 +/- 0.6 microM), than that on the normal human liver HL-7702 cells (IC(50): 9.1 +/- 0.4 microM). Of note, using tubulin polymerization assay, western blot and immuofluorescence analyses, XN0502 was showed to inhibit microtubule assembly at both molecular and cellular levels in A549 cells. Further studies indicated that XN0502 induced time- and dose-dependent G2/M arrest, accompanying with the reduction of CDC2/p34 expression and the downregulation of CDK7. The protein level alteration and the nuclear translocation of cyclinB1 were observed, denoting the M phase arrest in XN0502-treated cells. Moreover, XN0502 caused caspase-mediated apoptosis, as indicated by the cleavage of PARP, the reduction of procaspase-3 and procaspase-9, and the down-regulation of XIAP. Taken together, the current study demonstrates that the novel CA-4 analogue XN0502 is a promising anti-cancer agent with potent G2/M arrest- and apoptotic-inducing activities via targeting tubulin deserving further research and development, and helps provide data for exploiting new CA-4 analogues.

Piceatannol, a natural stilbene from grapes, induces G1 cell cycle arrest in androgen-insensitive DU145 human prostate cancer cells via the inhibition of CDK activity.

We have examined whether and by what mechanism piceatannol inhibits cell cycle progression in DU145 cells. The treatment of cells with piceatannol for 24h resulted in an increase in the percentage of cells in G1 phase and dose-dependent decreases in [(3)H]thymidine incorporation, as well as in protein levels of cyclin A, cyclin D1, and cyclin-dependent kinase (CDK)2 and CDK4. Piceatannol exerted no effect on the levels of p21(WAF1/CIP1) or p27(KIP1). Piceatannol reduced CDK4 and CDK2 activity. These results indicate that delaying G1 cell cycle progression contributes to the piceatannol-mediated inhibition of DU145 cell growth, which may be mediated via the inhibition of CDK activity.

Heat-processed neoginseng, KG-135, down-regulates G1 Cyclin-dependent kinase through the proteasome-mediated pathway in HeLa cells.

High temperature heat treatment of ginseng (Panax ginseng, C.A. Meyer) generates KG-135 (heat-processed neoginseng) which contains a mixture of three major ginseng saponins, ginsenosides Rk1, Rg3 and Rg5. Ginsenosides, particularly of the diol-type including Rk1, Rg3 and Rg5, have been shown to induce cell growth arrest in various cell types of human cancer. Herein, we report that KG-135 is able to arrest the cell cycle in human cervix adenocarcinoma HeLa cells. KG-135 arrests cells at the G1 phase of the cell cycle with an IC50 value of 69 microg/ml. The G1 phase arrest is associated with down-regulation of Cyclin D1/Cdk4 and Cyclin B1/Cdc2 activities in cells after treatment with KG-135. Furthermore, down-regulation of G1 Cyclin-dependent kinase activities is kinetically well related to the decreased intracellular protein levels of these kinases. In addition, the decrease in the levels of Cyclin D1/Cdk4 and Cyclin B1, but not of Cdc2, is similarly prevented by co-treatment of cells with MG-132, a potent proteasome inhibitor. Thus, the KG-135-induced arrest of the cell cycle at G1 phase in HeLa cells represents a novel mechanism that involves proteasome-mediated degradation of the Cyclins (Cyclin D1 and B1) and Cdk4 proteins.

Cyclin C and cyclin dependent kinases 1, 2 and 3 in thrombin-induced neuronal cell cycle progression and apoptosis.

The extent to which neurons proceed into the cell cycle and the mechanisms whereby cell cycle re-entry leads to apoptosis vary in response to agonists. We previously showed upregulation of early G1 regulators in thrombin-treated neurons yet neurons did not proceed to S phase but to apoptosis. The objective of this study is to explore mechanisms which might prevent S phase entry and promote apoptosis in thrombin-treated neurons. Cultured rat brain neurons are exposed to thrombin (200nM) for 30min to 4.5h and the expression of cyclin C, cyclin dependent kinases (cdk1, cdk2, cdk3, cdk8) and the cell cycle inhibitor p27 assessed. Our data show a simultaneous decrease of both cyclin C and cdk3 proteins soon after thrombin treatment. The decrease in cyclin C also correlates with decreases in cdk1 and cdk2, at both mRNA and protein levels. There is no change in expression of cdk8 or the cell cycle inhibitor p27 in response to thrombin treatment. These results suggest that decreases in G1-S regulators cyclin C and cdks 3, cdk2 and cdk1 in response to thrombin could make conditions unfavorable for S phase entry and favor neuronal apoptosis.

Oroxylin A induces G2/M phase cell-cycle arrest via inhibiting Cdk7-mediated expression of Cdc2/p34 in human gastric carcinoma BGC-823 cells.

We reported previously that oroxylin A, a natural product isolated from Scutellariae Radix, was a potent apoptosis inducer of human hepatoma HepG2 cells. In this study, cell-cycle arrest of BGC-823 human gastric carcinoma cells caused by oroxylin A has been investigated. Based on our 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay and flow cytometric analysis, treatment of BGC-823 cells with growth suppressive concentrations of oroxylin A caused an irreversible arrest in the G2/M phase of the cell cycle. Western blot analysis demonstrated that oroxylin A-induced cell-cycle arrest in BGC-823 cells was associated with a significant decrease in cdc2/p34, cyclin B1 and cyclin A expression. In addition, oroxylin A-treated cells decreased the expression of Cdk7, which was responsible for the low expression of M phase promoting factor (cyclin B1/Cdc2). The results suggested that oroxylin A induced G2/M phase cell-cycle arrest via inhibiting Cdk7-mediated expression of Cdc2/p34 in human gastric carcinoma BGC-823 cells.

Treatment with proteasome inhibitor MG132 during cloning improves survival and pronuclear number of reconstructed rat embryos.

In several mammalian species including rats, successfully cloned animals have been generated using somatic cell nuclear transfer (SCNT). However, in the case of rats, additional treatment with MG132, a proteasome inhibitor, before enucleation of oocytes seems to be required for successful cloning because ovulated rat oocytes are spontaneously activated, and hence, their suppression is the key to successful cloning. A previous study on rats demonstrated that matured oocytes potentially possess lower cytostatic factor (CSF) activity compared to mouse oocytes, resulting in a low incidence of premature chromosome condensation in the reconstructed embryos after SCNT. It is known that mice having more than two pronuclei are generally observed in nuclear-transferred oocytes after induction of premature chromosome condensation, which implies successful reprogramming. This leads us to the hypothesis that MG132 treatment affects not only the inhibition of spontaneous activation but also the reprogramming and developmental ability of reconstructed rat embryos. If so, prolonged MG132 treatment during and/or after SCNT may further improve the survivability. However, the effect of MG132 treatment on reconstructed embryos after SCNT has been very limited in rats and other species. We show here that prolonged MG132 treatment during and after SCNT improves survival and the number of pronuclei in reconstructed rat embryos after activation. These reconstructed embryos treated before, during, and after SCNT showed significantly higher p34(cdc2) kinase activity involving CSF activity compared to that of the control embryos. On the other hand, p34(cdc2) kinase activity was not recovered in nuclear-transferred oocytes without MG132, which suggested that the enucleation had detrimental effects on the development of reconstructed oocytes. Taken together, MG132 treatment during SCNT increases survival and pronuclear numbers in reconstructed rat embryos via maintenance of high CSF activity. The data suggest that MG132 treatment is indispensable for at least rat SCNT.

Cocaine regulates MEF2 to control synaptic and behavioral plasticity.

Repeated exposure to cocaine causes sensitized behavioral responses and increased dendritic spines on medium spiny neurons of the nucleus accumbens (NAc). We find that cocaine regulates myocyte enhancer factor 2 (MEF2) transcription factors to control these two processes in vivo. Cocaine suppresses striatal MEF2 activity in part through a mechanism involving cAMP, the regulator of calmodulin signaling (RCS), and calcineurin. We show that reducing MEF2 activity in the NAc in vivo is required for the cocaine-induced increases in dendritic spine density. Surprisingly, we find that increasing MEF2 activity in the NAc, which blocks the cocaine-induced increase in dendritic spine density, enhances sensitized behavioral responses to cocaine. Together, our findings implicate MEF2 as a key regulator of structural synapse plasticity and sensitized responses to cocaine and suggest that reducing MEF2 activity (and increasing spine density) in NAc may be a compensatory mechanism to limit long-lasting maladaptive behavioral responses to cocaine.

OSU03012 activates Erk1/2 and Cdks leading to the accumulation of cells in the S-phase and apoptosis.

OSU03012, a Celecoxib derivative, has been shown to inhibit proliferation and induce apoptosis in human cancer cell lines. However, its underlying mechanisms are not completely understood. In our study, the relationship between cell cycle inhibition and apoptosis induced by OSU03012 was investigated in human oral cancer cell lines. In the premalignant and malignant cell lines, OSU03012-induced growth inhibition, S-phase arrest, and apoptosis were accompanied by a marked increase in the activity of Erk1/2 and Cdk2/cyclin A. Inhibition of Cdks by roscovitine partially blocked OSU03012-induced growth inhibition and apoptosis. Although the activity of cdc2/cyclin B was reduced, expression of constructively active cdc2AF did not reverse OSU03012-induced S-phase arrest. When Erk1/2 was inhibited by U0126 before addition of OSU03012, growth inhibition and apoptosis induced by OSU03012 were attenuated. The levels of the Cdk2/cyclin A were reduced and cells accumulated in the G(0)/G(1) phase. When cells were allowed to accumulate in S-phase before addition of U0126, apoptosis also was attenuated suggesting that Erk1/2 is required for both progression of cells into the S-phase and apoptosis. Expression of constructively active MEK enhanced OSU03012-induced apoptosis. OSU03012 selectively inhibited the proliferation in premalignant and malignant, but not normal human oral cell lines. In conclusion, we show that OSU03012 has potent anti-proliferative and apoptotic activity against premalignant and malignant human oral cells through activation of Erk1/2, and Cdks. OSU0312 may provide unique opportunities for cancer prevention and sensitization of cancer cells to S-phase modalities.

Pyrogallol inhibits the growth of human lung cancer Calu-6 cells via arresting the cell cycle arrest.

Pyrogallol (PG) is a polyphenol compound and has been known to be an O(2)(.-) generator. We evaluated the effects of PG on the growth of human pulmonary adenocarcinoma Calu-6 cells in relation to the cell cycle. DNA flow cytometric analysis indicated that PG induced a G2 phase arrest of the cell cycle in Calu-6 cells at 72 h. PG down-regulated the expression of CDKI (p27), CDK2, CDK4 and CDK6 as well as cyclin D1, and increased cyclin A and cyclin B1 proteins. In addition, O(2)(.-) levels were significantly increased in PG-treated cells. Treatment with catalase rescued Calu-6 cells from PG-induced apoptosis, and also prevented the growth inhibition as well as a G2 phase arrest by PG, which were accompanied with the down-regulation of O(2)(.-) levels. In conclusion, PG inhibited the growth of Calu-6 cells by inducing the cell cycle arrest, accompanied with an increase in O(2)(.-) levels.