Thromboxane A2 Receptor Inhibition Suppresses Multiple Myeloma Cell Proliferation by Inducing p38/c-Jun N-terminal Kinase (JNK) Mitogen-activated Protein Kinase (MAPK)-mediated G2/M Progression Delay and Cell Apoptosis.

Multiple myeloma (MM) is a plasma cell malignancy without effective therapeutics. Thromboxane A2 (TxA2)/TxA2 receptor (T prostanoid receptor (TP)) modulates the progression of some carcinomas; however, its effects on MM cell proliferation remain unclear. In this study, we evaluated cyclooxygenase (COX) enzymes and downstream prostaglandin profiles in human myeloma cell lines RPMI-8226 and U-266 and analyzed the effects of COX-1/-2 inhibitors SC-560 and NS-398 on MM cell proliferation. Our observations implicate COX-2 as being involved in modulating cell proliferation. We further incubated MM cells with prostaglandin receptor antagonists or agonists and found that only the TP antagonist, SQ29548, suppressed MM cell proliferation. TP silencing and the TP agonist, U46619, further confirmed this finding. Moreover, SQ29548 and TP silencing promoted MM cell G2/M phase delay accompanied by reducing cyclin B1/cyclin-dependent kinase-1 (CDK1) mRNA and protein expression. Notably, cyclin B1 overexpression rescued MM cells from G2/M arrest. We also found that the TP agonist activated JNK and p38 MAPK phosphorylation, and inhibitors of JNK and p38 MAPK depressed U46619-induced proliferation and cyclin B1/CDK1 protein expression. In addition, SQ29548 and TP silencing led to the MM cell apoptotic rate increasing with improving caspase 3 activity. The knockdown of caspase 3 reversed the apoptotic rate. Taken together, our results suggest that TxA2/TP promotes MM cell proliferation by reducing cell delay at G2/M phase via elevating p38 MAPK/JNK-mediated cyclin B1/CDK1 expression and hindering cell apoptosis. The TP inhibitor has potential as a novel agent to target kinase cascades for MM therapy.

[Increased manganese superoxide dismutase and cyclin B1 expression in carnosine-induced inhibition of glioblastoma cell proliferation].

Carnosine is an endogenous dipeptide with antiproliferative properties. Here we show that carnosine selectively inhibits proliferation of human glioblastoma cells (U-118-MG) compared to breast (MB231) and oral (Cal27 and FaDu) cancer cells. Carnosine-induced inhibition of U-118-MG proliferation is associated with a significant: decrease in cellular reactive oxygen species levels, increase in manganese superoxide dismutase (MnSOD) and increase in cyclin B1 expression resulting in G2-block. We conclude that the antiproliferative property of carnosine is due to its ability to enhance MnSOD and cyclin B1 expression. These results will be of significance to the potential application of carnosine in brain cancer therapy.

Lentivirus­delivered nemo­like kinase small interfering RNA inhibits laryngeal cancer cell proliferation in vitro.

Laryngeal squamous cell carcinoma is the most common form of head and neck squamous cell carcinoma. Multiple approaches have been applied to treat this type of cancer; however, no significant improvement in survival rate has been achieved. In the present study, the role of nemo­like kinase (NLK) in human laryngeal carcinoma Hep­2 cells was investigated. NLK has been identified as an important regulator of cell growth, patterning and cell death in a variety of organisms. Lentivirus­mediated­shRNA was employed to silence endogenous NLK expression. Downregulation of the expression of NLK following lentivirus infection was confirmed using reverse transcription quantitative polymerase chain reaction and western blot analysis. The effects of NLK downregulation on Hep­2 cell proliferation and cell cycle progression were analyzed using an MTT assay and flow cytometry, respectively. Downregulation of NLK also inhibited tumorigenesis and regulated the expression of cell cycle protein expression levels. Therefore, it was hypothesized that NLK is necessary for cell survival and tumorigenesis in laryngeal cancer cells. Furthermore, the absence of NLK may lead to cancer cell death. Collectively, the results of the present study demonstrated that the lentivirus­mediated targeted disruption of NLK may be a promising therapeutic method for the treatment of laryngeal cancer.

Tubulin-binding agents down-regulate matrix metalloproteinase-2 and -9 in human hormone-refractory prostate cancer cells ­ a critical role of Cdk1 in mitotic entry.

Tubulin is an important target for anticancer therapy. Taxanes and vinca alkaloids are two groups of tubulin-binding agents in cancer chemotherapy. Besides tubulin binding, these groups of agents can also down-regulate protein levels of matrix metalloproteinase (MMP)-2 and -9, two important cancer-associated zinc-dependent endopeptidases in invasion and metastasis. However, the mechanism of action waits to be explored. In this study, protein levels but not mRNA expressions of MMP-2 and -9 were down-regulated by paclitaxel (a microtubule-stabilization agent), vincristine and evodiamine (two tubulin-depolymerization agents). These agents induced an increase of protein expression of cyclin B1, MPM2 (mitosis-specific phosphoprotein) and polo-like kinase (PLK) 1 phosphorylation. The data showed a negative relationship between the levels of mitotic proteins and MMP-2 and -9 expressions. MG132 (a specific cell-permeable proteasome inhibitor) blocked mitotic entry and arrested cell cycle at G2 phase, preventing down-regulation of MMP-2 and -9. Cell cycle synchronization experiments by thymidine block or nocodazole treatment showed that mitotic exit inhibited the down-regulation of MMP-2 and -9, confirming negative relationship between cell mitosis and protein levels of MMP-2 and -9 expressions. Cyclin-dependent kinase (Cdk) 1 is a key kinase in mitotic entry. Knockdown of Cdk1 almost completely inhibited the down-regulation of MMP-2 and -9 induced by tubulin-binding agents. In conclusion, the data suggest that mitotic entry and Cdk1 plays a central role in down-regulation of MMP-2 and -9 protein expressions. Tubulin-binding agents cause mitotic arrest and Cdk1 activation, which may contribute largely to the down-regulation of both MMP-2 and -9 expressions.

Preparation of morusin from Ramulus mori and its effects on mice with transplanted H22 hepatocarcinoma.

In this study, we used branches Ramulus mori from cultivated mulberry Husang-32 (Morus multicaulis Perry) as the experimental material and anhydrous ethanol as the extraction solution to obtain the crude extract from the branch bark. The ethanolic extract was successively purified through a macroporous resin, Sephadex LH-20, and semipreparative reversed-phase high-performance liquid chromatography (RP-HPLC). The high-purity monomer was identified as morusin by HPLC with diode array detection (HPLC-DAD) and its UV spectrum. The contents of morusin exhibited almost no difference between the root and branch bark in Husang-32, and morusin was not detected in the leaves. Morusin is able to inhibit the tumor growth of transplanted H22 hepatocarcinoma in mice and has no side effects. The fluorescence quantitative real-time PCR (qRT-PCR) results indicate that morusin has a marked inhibitory effect on liver cancer cells through a mechanism that may be related increases in the expression of p53, Survivin, CyclinB1, and Caspase-3 and a decrease in NF-κ B gene expression. The influence of this compound is more apparent in the Caspase-3 and the NF-κ B genes.