MK1775, a selective Wee1 inhibitor, shows single-agent antitumor activity against sarcoma cells.

Wee1 is a critical component of the G(2)-M cell-cycle checkpoint control and mediates cell-cycle arrest by regulating the phosphorylation of CDC2. Inhibition of Wee1 by a selective small molecule inhibitor MK1775 can abrogate G(2)-M checkpoint, resulting in premature mitotic entry and cell death. MK1775 has recently been tested in preclinical and clinical studies of human carcinoma to enhance the cytotoxic effect of DNA-damaging agents. However, its role in mesenchymal tumors, especially as a single agent, has not been explored. Here, we studied the cytotoxic effect of MK1775 in various sarcoma cell lines and patient-derived tumor explants ex vivo. Our data show that MK1775 treatment at clinically relevant concentrations leads to unscheduled entry into mitosis and initiation of apoptotic cell death in all sarcomas tested. In MK1775-treated cells, CDC2 activity was enhanced, as determined by decreased inhibitory phosphorylation of tyrosine-15 residue and increased expression of phosphorylated histone H3, a marker of mitotic entry. The cytotoxic effect of Wee1 inhibition on sarcoma cells seems to be independent of p53 status as all sarcoma cell lines with different p53 mutation were highly sensitive to MK1775 treatment. Finally, in patient-derived sarcoma samples, we showed that MK1775 as a single agent causes significant apoptotic cell death, suggesting that Wee1 inhibition may represent a novel approach in the treatment of sarcomas.

The cyclin-dependent kinase inhibitor SCH 727965 (dinacliclib) induces the apoptosis of osteosarcoma cells.

Although rare, osteosarcoma is an aggressive cancer that often metastasizes to the lungs. Toward the goal of developing new treatment options for osteosarcoma, we show that the cyclin-dependent kinase (CDK) inhibitor SCH 727965 (SCH) induces the apoptosis of several osteosarcoma cell lines including those resistant to doxorubicin and dasatinib. Cell lines prepared in our laboratory from patients who had received adjuvant chemotherapy and explants derived from a human osteosarcoma xenograft in mice were also responsive to SCH. Apoptosis occurred at low nanomolar concentrations of SCH, as did CDK inhibition, and was p53-independent. SCH activated the mitochondrial pathway of apoptosis as evidenced by caspase-9 cleavage and accumulation of cytoplasmic cytochrome c. Amounts of the apoptotic proteins Bax and Bim increased in mitochondria, whereas amounts of the antiapoptotic proteins Mcl-1 and Bcl-x(L) declined. Osteosarcoma cells apoptosed when codepleted of CDK1 and CDK2 but not when depleted of other CDK combinations. We suggest that SCH triggers the apoptosis of osteosarcoma cells by inactivating CDK1 and CDK2 and that SCH may be useful for treatment of drug-resistant osteosarcomas. SCH also induced the apoptosis of other sarcoma types but not of normal quiescent osteoblasts or fibroblasts.

Histone deacetylase inhibitors downregulate checkpoint kinase 1 expression to induce cell death in non-small cell lung cancer cells.

BACKGROUND: Histone deacetylase inhibitors (HDACis) are promising anticancer drugs; however, the molecular mechanisms leading to HDACi-induced cell death have not been well understood and no clear mechanism of resistance has been elucidated to explain limited efficacy of HDACis in clinical trials. METHODS AND FINDINGS: Here, we show that protein levels of checkpoint kinase 1 (Chk1), which has a major role in G(2) cell cycle checkpoint regulation, was markedly reduced at the protein and transcriptional levels in lung cancer cells treated with pan-and selective HDACis LBH589, scriptaid, valproic acid, apicidin, and MS-275. In HDACi treated cells Chk1 function was impaired as determined by decreased inhibitory phosphorylation of cdc25c and its downstream target cdc2 and increased expression of cdc25A and phosphorylated histone H3, a marker of mitotic entry. In time course experiments, Chk1 downregulation occurred after HDACi treatment, preceding apoptosis. Ectopic expression of Chk1 overcame HDACi-induced cell death, and pretreating cells with the cdc2 inhibitor purvalanol A blocked entry into mitosis and prevented cell death by HDACis. Finally, pharmacological inhibition of Chk1 showed strong synergistic effect with LBH589 in lung cancer cells. CONCLUSIONS: These results define a pathway through which Chk1 inhibition can mediate HDACi-induced mitotic entry and cell death and suggest that Chk1 could be an early pharmacodynamic marker to assess HDACi efficacy in clinical samples.

Differential proinflammatory and angiogenesis-specific cytokine production in human pulmonary endothelial cells, HPMEC-ST1.6R infected with dengue-2 and dengue-3 virus.

In this study, the ability of dengue virus serotypes 2 (DENV-2) and 3 (DENV-3) to infect and induce increased production of proinflammatory cytokines in a pulmonary endothelial cell line (HPMEC-ST1.6R) was investigated. This cell line exhibits the major constitutive and inducible endothelial cell characteristics, as well as angiogenic response. DENV-2 and DENV-3 infection was confirmed by an observed cytopathic effect (CPE), as well as RT-PCR and immunofluorescence assays. Increases in Th-1 and Th-2 cytokines IL-4, IL-8, IL-6, IL-10, GM-CSF, INF-gamma, and tumor necrosis factor (TNF-alpha) within DENV-2- and DENV-3-infected cells were demonstrated using a microbead-based Bio-plex assay. Proinflammatory cytokine increases and the expression of a potent angiogenic inducer protein, VEGF were confirmed by dot-blot analysis using the TranSignal Human Angiogenesis Antibody Array. Dengue virus-infected HPMEC-ST1.6R cells exhibited an elongated cytoplasmic morphology, possibly representing a response to VEGF and activation of angiogenesis. The increased levels of Th-1 cytokines and VEGF in DENV-2 virus infected-HPMEC-ST1.6R could be distinguished from those infected by DENV-3. This suggests that cytokine patterns associated with DENV infections may be serotype and strain-specific. The experimental approaches described here could be developed further into a useful diagnostic tool for the characterization of dengue hemorrhagic fever cases, leading to enhancement of treatment therapy.