Effect Of Stabilized MK5 On Enhancing Apoptosis Of Hepatocellular Carcinoma Cells Induced By

Hepatocellular carcinoma (HCC) and pancreatic cancer are most common malignant tumors worldwide and frequently diagnosed at advanced stages and have a poor prognosis with a high mortality rate. Over the past decade, curative surgery has become the mainstay of treatment for HCC and pancreatic cancer and the first option for patients with early-stage tumors. However, many patients have been diagnosed with unresectable tumors at the time of initial diagnosis due to lack of early detection methods. Chemotherapy has proven to be an effective treatment modality especially for patients with inoperable tumors.Doxorubicin (DOX), an anthracycline-based anticancer drug, is widely used for HCC among various chemotherapeutic drugs. Recent studies have demonstrated that the response rate to DOX becomes lower and lower. Therefore, it is of great significance to search for novel molecular targets for developing more effective treatment methods.On the other hand, the5-year survival rate of pancreatic cancer was only5%. The pancreatic ductal adenocarcinoma accounts for75%-92%of the total pancreatic cancer and it is the focus in pancreatic cancer research. The clinical drug for pancreatic ductal adenocarcinoma treatment is comparatively few, and the effective drugs for other tumors are often difficult to work in pancreatic ductal adenocarcinoma.In the first section of this study, we describle a novel role of MK5in doxorubicin-induced hepatoma apoptosis. MAPKAPK5(MK5), also named as p38-regulated/activated protein kinase (PRAK), has been identified as a crucial mediator essential for both suppression and promotion of skin tumorigenesis, depending on the stage of cancer development. There is little evidence indicating that MK5is involved in HCC. To explore the expression of MK5in HCC tissues, we examined MK5expression in16paired HCC specimens by Western blot. MK5expression was upregulated in10out of16HCCs as assessed. To investigate the physiological role of MK5in hepatoma cells, we generated stable cell lines with overexpression or knockdown of MK5in HepG2and Hep3B cells which were used for further functional analysis. Stable overexpression or knockdown of MK5, respectively, enhanced or reduced hepatoma cell (HepG2and Hep3B) viability under treatment of doxorubicin rather than other chemotherapeutic drugs, such as CPT, VCR and TAX. We further showed that doxorubicin decreases MK5expression in a time-and concentration-dependent manner. We found that the MK5mRNA levels in cells treated with DOX were not change significantly, indicating that DOX downregulates MK5 expression at the posttranscriptional level. In contrary, degradation of MK5protein was induced in response to DOX via the26S proteasome. Moreover, overexpression of MK5attenuated doxorubicin-induced apoptosis and led to decreased cleavage of caspase-3and PARP, while knockdown of endogenous MK5sensitized cells to doxorubicin, which was coupled with increased cleavage of caspase-3and PARP.In the second section of this study, our findings provide a novel regulatory mechanism of BRSK2, an upregulated protein in PANC-1cell, through direct interaction with Jab1. Brain-specific kinase2(BRSK2) was classified as an AMP-activated protein kinase (AMPK)-related kinase and one of the substrates of LKB1. Early study in our lab found that BRSK2is critical for tolerance of PANC-1cell in glucose starvation and it is a potential target for pancreatic ductal adenocarcinoma treatment. However, the molecular regulatory mechanisms of BRSK2are incompletely understood. In this study, we isolated a novel BRSK2-interacting protein, c-Jun activation domain-binding protein-1(Jab1), which was reported to mediate degradation of multiple proteins and positively regulate cell cycle progression. GST pull-down and immunoprecipitation assays revealed the direct interaction between BRSK2and Jab1in vitro and in vivo, respectively. The co-localization between Jab1and BRSK2in the perinuclear region was observed. Intriguingly, Jab1promoted the ubiquitination and proteasome-dependent degradation of BRSK2. Silencing of endogenous Jab1increased the cellular BRSK2protein level. Consistent with this*BRSK2-mediated cell cycle arrest at the G2/M phase in mammalian cells was reversed by exogenous Jabl. Taken together, our findings provide a novel regulatory mechanism of BRSK2through direct interaction with Jab1.Taken together, our results firstly demonstrate that proteasomal degradation of MK5contributes to doxorubicin-induced apoptosis in hepatoma cells, providing novel insights into the molecular mechanism of drug resistance in hepatoma cells and a novel target for developing an optimal therapeutic strategy for HCC patients. In addition, we present novel findings showing that BRSK2directly interacts with Jab1in vitro and in vivo, suggesting a new clue for pancreatic ductal adenocarcinoma treatment.