Selective Activation of ZAK ß Expression by 3-Hydroxy-2-Phenylchromone Inhibits Human Osteosarcoma Cells and Triggers Apoptosis via JNK Activation.

Although various advancements in radical surgery and neoadjuvant chemotherapy have been developed in treating osteosarcoma (OS), their clinical prognosis remains poor. A synthetic chemical compound, 3-hydroxylflavone, that is reported to regulate ROS production is known to inhibit human bone osteosarcoma cells. However, its role and mechanism in human OS cells remains unclear. In this study, we have determined the potential of 3-Hydroxy-2-phenylchromone (3-HF) against OS using human osteosarcoma (HOS) cells. Our previous studies showed that Zipper sterile-alpha-motif kinase (ZAK), a kinase member of the MAP3K family, was involved in various cellular events such as cell proliferation and cell apoptosis, and encoded two transcriptional variants, ZAKα and ß. In this study, we show that 3-HF induces the expression of ZAK and thereby enhances cellular apoptosis. Using gain of function and loss of function studies, we have demonstrated that ZAK activation by 3-HF in OS cells is confined to a ZAKß form that presumably plays a leading role in triggering ZAKα expression, resulting in an aggravated cancer apoptosis. Our results also validate ZAKß as the predominant form of ZAK to drive the anticancer mechanism in HOS cells.

Fisetin activates Hippo pathway and JNK/ERK/AP-1 signaling to inhibit proliferation and induce apoptosis of human osteosarcoma cells via ZAK overexpression.

Osteosarcoma (OS) is a tumor entity that can cause a large number of cancer-related deaths. Although chemotherapy can decrease proliferation and increase apoptosis of human OS cells, the clinical prognosis remains poor. Fisetin is a flavonol found in fruits and vegetables and is reported to inhibit cell growth in numerous cancers. But the molecular mechanism underlying fisetin in human OS cells is not clear. It is known that sterile-alpha motif and leucine zipper containing kinase (ZAK), a kinase in the MAP3K family, is involved in various cell processes, including proliferation and apoptosis. In our lab, we have demonstrated that overexpression of ZAK can induce apoptosis in human OS cells. In the previous studies, MAP4K, the upstream of MAP3K, can act in parallel to MST1/2 to activate LATS1/2 in the Hippo pathway. Turning on the Hippo pathway can decrease proliferation and otherwise cause cell apoptosis in cancer cells. In this study, we found that fisetin can upregulate ZAK expression to induce the Hippo pathway and mediate the activation of JNK/ERK, the downstream of ZAK, to trigger cell apoptosis via AP-1 dependent manner in human OS cells. These findings reveal a novel molecular mechanism underlying fisetin effect on human OS cells.

Overexpression of ZAKß in human osteosarcoma cells enhances ZAKα expression, resulting in a synergistic apoptotic effect.

ZAK is a novel mixed lineage kinase-like protein that contains a leucine-zipper and a sterile-alpha motif as a protein-protein interaction domain, and it is located in the cytoplasm. There are 2 alternatively spliced forms of ZAK: ZAKα and ZAKß. Previous studies showed that ZAKα is involved in various cell processes, including cell proliferation, cell differentiation, and cardiac hypertrophy, but the molecular mechanism of ZAKß is not yet known. In a recent study in our laboratory, we found that ZAKß can ameliorate the apoptotic effect induced by ZAKα in H9c2 cells. We further hypothesized that ZAKß could also improve the apoptotic effect induced by ZAKα in human osteosarcoma cells. The results of this study show that ZAKß can induce apoptosis and decrease cell viability similar to the effects of ZAKα. Interestingly, our ZAKα-specific inhibitor assay shows that the expression of ZAKß is highly dependent on ZAKα expression. However, ZAKß expression effectively induces ZAKα expression and results in synergistic enhancement of apoptosis in human osteosarcoma cells. Furthermore, co-immunoprecipitation results revealed that ZAKα can directly interact with ZAKß, and this interaction may contribute to the enhanced apoptotic effects. SIGNIFICANCE OF THE STUDY: ZAK is a mixed lineage kinase involved in cell differentiation, proliferation, and hypertrophic growth. ZAKα isoform of ZAK is associated with tumorigenesis, but the function of ZAKß is not yet known. In H9c2 cells, ZAKß was found to ameliorate the apoptotic effect induced by ZAKα. However, in osteosarcoma cells, ZAKß elevates the apoptotic effect induced by ZAKα. In this study, we show that similar to ZAKα, the ZAKß induces apoptosis and decreases cell viability. Interestingly, the expression of ZAKß is dependent on ZAKα expression, and ZAKß further enhances ZAKα expression and results in synergistic enhancement of apoptosis in osteosarcoma cells.

Doxorubicin induces ZAKα overexpression with a subsequent enhancement of apoptosis and attenuation of survivability in human osteosarcoma cells.

Human osteosarcoma (OS) is a malignant cancer of the bone. It exhibits a characteristic malignant osteoblastic transformation and produces a diseased osteoid. A previous study demonstrated that doxorubicin (DOX) chemotherapy decreases human OS cell proliferation and might enhance the relative RNA expression of ZAK. However, the impact of ZAKα overexpression on the OS cell proliferation that is inhibited by DOX and the molecular mechanism underlying this effect are not yet known. ZAK is a protein kinase of the MAPKKK family and functions to promote apoptosis. In our study, we found that ZAKα overexpression induced an apoptotic effect in human OS cells. Treatment of human OS cells with DOX enhanced ZAKα expression and decreased cancer cell viability while increasing apoptosis of human OS cells. In the meantime, suppression of ZAKα expression using shRNA and inhibitor D1771 both suppressed the DOX therapeutic effect. These findings reveal a novel molecular mechanism underlying the DOX effect on human OS cells. Taken together, our findings demonstrate that ZAKα enhances the apoptotic effect and decreases cell viability in DOX-treated human OS cells.

CDK2 phosphorylation regulates the protein stability of KLF10 by interfering with binding of the E3 ligase SIAH1.

Downregulation of multiple cell cycle-regulatory molecules is a dominant event in TGF-ß1-mediated growth inhibition of human carcinoma cells. It is known that KLF10 mimics the anti-proliferative and apoptotic effects that TGF-ß1 has on epithelial cell growth and the growth of various tumor cells; based on these findings it is considered as a tumor suppressor. KLF10 protein expression is tightly associated with cell cycle-dependent events. However, the regulatory mechanism and its biological meaning have not been identified. In this study, we have demonstrated that KLF10 is a substrate of CDK2/cyclin E and can be phosphorylated. We also have shown that KLF10 efficiently binds to CDK2, while binding much less to CDK4, and displaying no binding to Cdk6. Using mass spectrometry, site direct mutagenesis, in vitro kinase assays and depletion assays, we have established that CDK2 phosphorylates Ser206, which subsequently affects the steady state level of KLF10 in cells. Our studies have also proved that CDK2 up-regulates the protein level of KLF10 through reducing its association with SIAH1, a KLF10 E3-ubiqutin ligase involved in proteasomal degradation. Taken all together, these findings indicate that CDK2-dependent phosphorylation regulates KLF10 stability and that this affects the role of KLF10 in cell.

ZAKß antagonizes and ameliorates the cardiac hypertrophic and apoptotic effects induced by ZAKα.

ZAK (sterile alpha motif and leucine zipper containing kinase AZK), a serine/threonine kinase with multiple biochemical functions, has been associated with various cell processes, including cell proliferation, cell differentiation, and cardiac hypertrophy. In our previous reports, we found that the activation of ZAKα signaling was critical for cardiac hypertrophy. In this study, we show that the expression of ZAKα activated apoptosis through both a FAS-dependent pathway and a mitochondria-dependent pathway by subsequently inducing caspase-3. ZAKß, an isoform of ZAKα, is dramatically expressed during cardiac hypertrophy and apoptosis. The interaction between ZAKα and ZAKß was demonstrated here using immunoprecipitation. The results show that ZAKß has the ability to diminish the expression level of ZAKα. These findings reveal an inherent regulatory role of ZAKß to antagonize ZAKα and to subsequently downregulate the cardiac hypertrophy and apoptosis induced by ZAKα.

Kefir peptides promote osteogenic differentiation to enhance bone fracture healing in rats.

AIMS: Fractures are the result of fragile bone structures after trauma caused by direct or indirect external impact or strong muscular contraction. Most fracture patients undergo surgical fixation to accelerate the healing process and restore the function of mutilated bone. Promoting the healing process remains an important issue for the treatment of bone fractures. Our previous studies demonstrated the remarkable bone-protective effects of kefir peptides (KPs) in ovariectomized rats and mice. In this study, we further evaluate the efficacy of KPs on fracture healing using a rat model of femoral fracture. MAIN METHODS: Fifteen 8-week-old male Sprague Dawley (SD) rats were divided into the sham, mock, and KPs groups, in which the mock and the KPs groups underwent femur-fracture surgery with nail fixation, while the sham group underwent a sham operation. The next day, rats were orally administered with daily 400 mg/kg of KPs (KPs group) or distilled water (sham and mock groups) for four weeks. X-ray imaging, histochemical staining and serum osteogenic markers were applied for fracture healing evaluation. In vitro, mouse bone marrow mesenchymal stem cells (BMMSCs) and MC3T3-E1 line were subjected to osteoblast differentiation in the presence of KPs and compared with no KPs treatment. KEY FINDINGS: The results demonstrated that KPs treatment improved the progression of the fracture healing process (p < 0.05) and significantly increased the expressions of Col1a1, Alp, Spp1, Vegfa and Cox2 mRNA in the femurs of the KPs-treated fractured rats compared to those of the mock-treated fracture rats. In vitro, KPs treatment promoted bone regeneration factor (Col1a1, Alp, M-csf and Phospho1) expression in MC3T3-E1-derived osteoblast cultures (on Day 3) and enhanced osteogenic differentiation and mineralization in BMMSC-derived osteoblast cultures (on Day 17 and Day 21). SIGNIFICANCE: This is the first study to show that KPs can help with fracture healing by promoting osteogenic differentiation, and it also suggests that KPs can be used as a nutritional supplement to accelerate fracture healing.

A novel role for ß2-microglobulin: a precursor of antibacterial chemokine in respiratory epithelial cells.

We analyzed a panel of cationic molecules secreted in the culture medium of human respiratory epithelial cells (REC) upon activation by IL-1ß and different pathogen-associated molecular patterns. A 9 kDa fragment derived from ß2-microglobulin (B2M) was identified and named shed 9 kDa B2M (sB2M-9). The primary structure of sB2M-9 was revealed to increase its pI value that potentially could play an important role in innate defense. sB2M-9 exhibits antibacterial activity against Gram positive Staphylococcus aureus (SA) but not against Gram negative Klebsiella pneumonia (KP). Upon its binding to SA, sB2M-9 induces clumps, a phenomenon not observed with B2M. Migration of THP-1 monocytes exposed to SA clumps was significantly greater than that to SA without clumps. sB2M-9 binds to SA, more likely as a chemokine, to facilitate THP-1 migration. As a whole, we demonstrated that REC release a novel chemokine with antibacterial activity that is shed from B2M to facilitate THP-1 migration.