Knockdown of HURP inhibits the proliferation of hepacellular carcinoma cells via downregulation of gankyrin and accumulation of p53.

We determined earlier that the hepatoma upregulated protein (HURP) is overexpressed in hepatocellular carcinoma (HCC), but the role of this protein during cancer development and progression remains unknown. Here, we observed that the overexpression of HURP in HEK293 cells promoted the ubiquitination of p53 and its degradation by the proteasome. In contrast, HURP knockdown using short-hairpin RNA reversed these effects. Knockdown of HURP promoted the accumulation of p53 in SK-Hep-1 cells (p53+/-), and these cells showed reduced proliferation, while the p53-mutant Mahlavu cells were not affected. HURP knockdown did not affect the proliferation of H1299 lung carcinoma cells and Hep3B HCC cells which lack p53. Knockdown of HURP also sensitized SK-Hep-1 cells to cisplatin. On the other hand, the expression of exogenous p53 in H1299 and Hep3B cells was decreased following overexpression of HURP, and these cells showed decreased sensitivity to cisplatin-induced apoptosis. Importantly, overexpression of HURP promoted the proliferation of HEK293 cells in an anchorage-independent manner, and inoculation of SK-Hep-1 cancer cells that expressed short-hairpin RNA to knockdown HURP resulted in smaller tumors in nude mice. Gankyrin, a positive regulator of the E3 ubiquitin ligase MDM2, was found to be upregulated following HURP expression, and gankyrin knockdown decreased the HURP-mediated downregulation of p53. Notably, we detected a positive correlation between elevated HURP and gankyrin protein levels in HCC patients (r(2) = 0.778; N = 9). Taken together, these results indicate that HURP represents an oncogene that may play a role in HCC progression and chemoresistance.

Latent membrane protein 1 of Epstein-Barr virus sensitizes cancer cells to cisplatin by enhancing NF-κB p50 homodimer formation and downregulating NAPA expression.

Expression of the oncogenic latent membrane protein 1 (LMP1) of Epstein-Barr virus is involved in the pathogenesis of nasopharyngeal carcinoma (NPC) and lymphoma. In previous studies, we found that expression of LMP1 was sufficient to transform BALB/c-3T3 cells. In contrast, other studies have shown that LMP1 induces apoptosis in a NF-κB-dependent manner and also inhibits the growth of tumors in mice, thereby indicating that LMP1 may produce various biological effects depending on the biological and cellular context. Still, the mechanism underlying the pro-apoptotic activity of LMP1 remains unclear. In the present study, we found that LMP1 inhibits the expression of NAPA, an endoplasmic reticulum SNARE protein that possesses anti-apoptotic properties against the DNA-damaging drug cisplatin. Accordingly, LMP1-transformed BALB/c-3T3 cells were sensitized to cisplatin-induced apoptosis, whereas no sensitization effect was noted following treatment with the mitotic spindle-damaging drugs vincristine and taxol. Knockdown of LMP1 with antisense oligonucleotides restored NAPA protein level and rendered the cells resistant to cisplatin. Similarly, overexpression of NAPA reduced the effect of LMP1 and induced resistance to cisplatin. LMP1 was shown to upregulate the NF-κB subunit p50, leading to formation of p50 homodimers on the NAPA promoter. These findings suggest that the viral protein LMP1 may sensitize cancer cells to cisplatin chemotherapy by downregulating NAPA and by enhancing the formation of p50 homodimers which in turn inhibit the expression of NF-κB regulated anti-apoptotic genes. These findings provide an explanatory mechanism for the pro-apoptotic activity of LMP1 as well as new therapeutic targets to control tumor growth.

The tyrosine kinase inhibitor sorafenib sensitizes hepatocellular carcinoma cells to taxol by suppressing the HURP protein.

The hepatoma upregulated protein (HURP) represents a putative oncogene that is overexpressed in many human cancers, especially hepatocellular carcinoma (HCC). HURP plays an important role during mitotic spindle formation, a process that is targeted by various anti-cancer drugs like taxol. However, the role of HURP during the establishment of taxol chemoresistance in HCC remains unclear. In this study, we observed that high HURP protein level correlates with taxol resistance in HCC cells. Following HURP knockdown, HCC cells show a more sensitive response to taxol treatment. Notably, sorafenib, a tyrosine kinase inhibitor approved for the treatment of HCC, inhibits HURP expression primarily at the transcriptional level and sensitizes HCC cells to sub-lethal doses of taxol. By using real-time PCR and chromatin immunoprecipitation assays, we observed that the NF-κB family member c-Rel represents a putative transcription factor that activates HURP gene expression. In addition, the inhibitory effect of sorafenib on HURP expression was attributed to a reduced translation and nuclear translocation of c-Rel. Accordingly, downregulation of c-Rel using short-hairpin RNA was shown to reduce HURP protein level and enhance taxol-induced cell death. Taken together, our results indicate that HURP acts as a novel survival protein that protects HCC cells against taxol-induced cell death. In addition, the regulation of HURP gene expression by NF-κB signaling appears to be critical for the response of HCC cells to taxol.

Hepatitis B virus X protein prevents apoptosis of hepatocellular carcinoma cells by upregulating SATB1 and HURP expression.

Protein X from hepatitis B virus (HBV) appears to play a critical role in the development of hepatocellular carcinoma (HCC). The hepatoma upregulated protein (HURP) is also upregulated in a majority of HCC cases, therefore suggesting that HURP represents an oncogene. In this study, we describe a link between the viral protein HBx, HURP, and the establishment of cisplatin chemoresistance in HCC cells. Hep3B cells which express HBx displayed increased levels of HURP mRNA and protein, and showed resistance to cisplatin-induced apoptosis. Knockdown of HURP in HBx-expressing cells reversed this effect and sensitized Hep3B cells to cisplatin. Interestingly, SATB1, a global gene regulator which is often overexpressed in malignant breast cancer, was also induced following expression of HBx. The anti-apoptotic effect of HBx was shown to require activation of the p38/MAPK pathway in Hep3B cells. In addition, the expression of survivin, an anti-apoptotic protein, was also upregulated by HBx in an HURP-dependent manner. Taken together, these results indicate that HBx activates the expression of HURP via the p38/MAPK pathway and the SATB1 protein, culminating with the accumulation of the anti-apoptotic protein survivin. Our findings illustrate the role of the viral protein HBx in preventing apoptosis during cancer progression and establishment of chemoresistance.

Emodin has cytotoxic and protective effects in rat C6 glioma cells: roles of Mdr1a and nuclear factor kappaB in cell survival.

1,3,8-Trihydroxy-6-methylanthaquinone (emodin) is recognized as an antiproliferative compound. In the present study, however, we show that emodin has both toxic and survival effects in glioma cells and that the survival effects involve Mdr1a. Emodin inhibited the proliferation and induced apoptosis of C6 cells in a 12-h treatment, but C6 cells survived a 72-h drug treatment, indicating resistance to emodin. Emodin-induced apoptosis was reduced by inhibition of the expression and activation of apoptosis-associated proteins including p53, Bax, Bcl-2, Fas, and caspase-3. C6 cells could express antioxidant proteins (superoxide dismutase and catalase) to decrease reactive oxygen species-induced cytotoxicity of emodin and overexpress multidrug resistance genes (Mdr1a, MRP2, MRP3, and MRP6) to decrease the intracellular accumulation of emodin. Electrophoretic mobility shift analysis showed that emodin decreased nuclear factor kappaB (NF-kappaB) expression in 24 h of treatment, but in 48 h, emodin increased NF-kappaB activity. A confocal microscope showed that emodin induced NF-kappaB translocation from cytoplasm to nuclei. C6 cells would activate the mitogen-activated protein kinase survival pathway and express the DNA repair gene (MGMT) and associated proteins (PARP and XRCC1) to recover the cell activity. C6 cells also expressed GRP78 to decrease emodin-induced endoplasmic reticulum (ER) stress that would cause apoptosis in C6 cells, and GRP78 inhibited the expression of GADD153 to enhance the expression of Bcl-2 that could balance the ER- and mitochondria-induced apoptosis of C6 cells.

Diallyl disulfide inhibits WEHI-3 leukemia cells in vivo.

Enhanced garlic (Allium sativum) consumption is closely related to reduced cancer incidence, as shown in epidemiological studies. Diallyl disulfide (DADS), a component of garlic, inhibits the proliferation of human blood, colon, lung and skin cancer cells. Although DADS had been reported to induce apoptosis in human leukemia HL-60 cells, there are no reports regarding whether or not it affects leukemia cells in vivo. Therefore, the present study is focused on the in vivo effects of DADS on WEHI-3 leukemia cells. The effects of DADS on murine WEHI-3 cells were initially examined, and the results indicated that DADS induced cytotoxicity and that this effect was dose-dependent. The effects of DADS on WEHI-3 in BALBIc mice were also examined, and the results indicated that DADS decreased the percentage of MAC-3 marker, indicating that differentiation of the precursor of macrophage and T cells was inhibited. The weights of liver and spleen were also measured, and the results indicated that DADS decreased the weight of these organs. An important characteristic of WEHI-3 leukemia is the enlarged spleen in mice after i.p. injection of WEHI-3 cells. Based on pathological examination, the function of DADS was observed in the liver and spleen of mice previously injected with WEHI-3 cells. Apparently, DADS affects WEHI-3 cells both in vitro and in vivo.