Effects of endoplasmic reticulum stress on the autophagy, apoptosis, and chemotherapy resistance of human breast cancer cells by regulating the PI3K/AKT/mTOR signaling pathway.

Nowadays, although chemotherapy is an established therapy for breast cancer, the molecular mechanisms of chemotherapy resistance in breast cancer remain poorly understood. This study aims to explore the effects of endoplasmic reticulum stress on autophagy, apoptosis, and chemotherapy resistance in human breast cancer cells by regulating PI3K/AKT/mTOR signaling pathway. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to detect the cell viability of six human breast cancer cell lines (MCF-7, ZR-75-30, T47D, MDA-MB-435s, MDA-MB-453, and MDA-MB-231) treated with tunicamycin (5 µM), after which MCF-7 cells were selected for further experiment. Then, MCF-7 cells were divided into the control (without any treatment), tunicamycin (8 µ), BEZ235 (5 µ), and tunicamycin + BEZ235 groups. Cell viability of each group was testified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Western blotting was applied to determine the expressions of endoplasmic reticulum stress and PI3K/AKT/mTOR pathway-related proteins and autophagy- and apoptosis-related proteins. Monodansylcadaverine and Annexin V-fluorescein isothiocyanate/propidium iodide staining were used for determination of cell autophagy and apoptosis. Furthermore, MCF-7 cells were divided into the control (without any treatment), tunicamycin (5 µM), cisplatin (16 µM), cisplatin (16 µM) + BEZ235 (5 µM), tunicamycin (5 µM) + cisplatin (16 µM), and tunicamycin (5 µM) + cisplatin (16 µM) + BEZ235 groups. Cell viability and apoptosis were also evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and Annexin V-fluorescein isothiocyanate/propidium iodide staining. In MCF-7 cells treated with tunicamycin, cell viability decreased significantly, but PEAK, eIF2, and CHOP were upregulated markedly and p-PI3K, p-AKT, and p-MTOR were downregulated in dose- and time-dependent manners. In the tunicamycin + BEZ235 group, the cell viability was lower and the apoptosis rate was higher than those of the control and monotherapy groups. Compared with the cisplatin group, the tunicamycin + cisplatin group showed a relatively higher growth inhibition rate; the growth inhibition rate substantially increased in the tunicamycin + cisplatin + BEZ235 group than the tunicamycin + cisplatin group. The apoptosis rate was highest in tunicamycin + cisplatin + BEZ235 group, followed by tunicamycin + cisplatin group and then cisplatin group. Our study provide evidence that endoplasmic reticulum stress activated by tunicamycin could promote breast cancer cell autophagy and apoptosis and enhance chemosensitivity of MCF-7 cells by inhibiting the PI3K/AKT/mTOR signaling pathway.

Stat3-siRNA inhibits the growth of gastric cancer in vitro and in vivo.

Gastric cancer remains one of the most prevalent and lethal malignancies in the world. Despite new advances in treatment and diagnosis, patients with advanced gastric cancer are still difficult to cure resulting in a high mortality rate and poor prognosis. Signal transducer and activator of transcription 3 (Stat3) is observed aberrant in multiple tumours, including gastric cancer. Stat3 overexpression was confirmed performing a vital role in tumorigenesis. In the present study, we constructed a pSi-Stat3 plasmid to silence Stat3 and investigated the effect of pSi-Stat3 on cell proliferation, apoptosis and cell cycle progression in gastric cancer cell line SGC-7901 and mice xenograft model. Downstream proteins of Stat3, including Cyclin-D1, Survivin and Bcl-2, were detected as well for the underlying mechanism exploration. It showed that pSi-Stat3 can effectively silence the expression of Stat3 and inhibits the growth of gastric tumour both in vitro and in vivo significantly via cell apoptosis and cell cycle shift induction. The findings suggest that Stat3 signal pathway might be a promising therapeutic target for tumour treatment, including gastric cancer.

Correlations of the expressions of c-Jun and Egr-1 proteins with clinicopathological features and prognosis of patients with nasopharyngeal carcinoma.

This study intended to explore the correlation of the expressions of c-Jun and Egr-1 proteins with clinicopathological features and prognosis of patients with nasopharyngeal carcinoma (NPC). From January 2008 to January 2011, 123 NPC patients and 59 patients with chronic rhinitis were enrolled in this study. Fresh NPC and normal nasopharynx tissue specimens were obtained during surgery. Immunohistochemistry (IHC) was adopted to determine the positive expressions of the c-Jun and Egr-1 proteins. A 5-year clinical follow-up was conducted on all NPC patients. The Kaplan-Meier survival curve and Cox regression model were used for survival analysis. Compared with normal nasopharynx tissues, c-Jun expression was up-regulated but Egr-1 expression was down-regulated in NPC tissues. NPC patients with stage T3-T4 or stage III-IV had higher positive rates of c-Jun expression than those with stage T1-T2 or stage I-II. However, the positive rates of Egr-1 expression was higher in patients with stage T1-T2 or stage III-IV than those with stage T3-T4 or stage I-II. The survival rate of NPC patients with high c-Jun expression was lower than those with low/negative c-Jun expression, while the survival rate of NPC patients with high Egr-1 expression was higher than those with low/negative Egr-1 expression. The Cox regression analysis revealed that stage T3-T4, high c-Jun expression, and low Egr-1 expression were risk factors for poor prognosis of NPC patients. In conclusion, our study suggests that the c-Jun and Egr-1 proteins can serve as novel potential biomarkers for the early diagnosis and prognosis prediction of NPC.

p38 MAPK signaling mediates mitochondrial apoptosis in cancer cells induced by oleanolic acid.

Oleanolic acid (OA) is a nutritional component widely distributed in various vegetables. Although it has been well recognized for decades that OA exerts certain anti-tumor activity by inducing mitochondria-dependent apoptosis, it is still unclear that what molecular signaling is responsible for this effect. In this study, we employed cancer cell lines, A549, BXPC-3, PANC-1 and U2OS to elucidate the molecular mechanisms underlying OA anti- tumor activity. We found that activation of MAPK pathways, including p-38 MAPK, JNK and ERK, was triggered by OA in both a dose and time-dependent fashion in all the tested cancer cells. Activation was accompanied by cleavage of caspases and PARP as well as cytochrome C release. SB203580 (p38 MAPK inhibitor), but not SP600125 (JNK inhibitor) and U0126 (ERK inhibitor), rescued the pro-apoptotic effect of OA on A549 and BXPC- 3 cells. OA induced p38 MAPK activation promoted mitochondrial translocation of Bax and Bim, and inhibited Bcl-2 function by enhancing their phosphorylation. OA can induce reactive oxygen species (ROS)-dependent ASK1 activation, and this event was indispensable for p38 MAPK-dependent apoptosis in cancer cells. In vivo, p38 MAPK knockdown A549 tumors proved resistant to the growth-inhibitory effect of OA. Collectively, we elucidated that activation of ROS/ASK1/p38 MAPK pathways is responsible for the apoptosis stimulated by OA in cancer cells. Our finding can contribute to a better understanding of molecular mechanisms underlying the antitumor activity of nutritional components.

The p38 MAPK inhibitor JLU1124 inhibits the inflammatory response induced by lipopolysaccharide through the MAPK-NF-κB pathway in RAW264.7 macrophages.

Our previous results showed that JLU1124 is a potent p38 mitogen-activated protein kinase (MAPK) inhibitor. Compared to the classic p38 MAPK inhibitor SB203580, JLU1124 inhibits p38 phosphorylation at low concentrations without cytotoxicity on cells. p38 MAPK is a known target for inflammation treatment. Thus, we became interested in whether JLU1124 has anti-inflammatory effects. We used LPS stimulated RAW264.7 macrophages as a model of inflammation to evaluate the anti-inflammatory effects of JLU1124. Our results showed that p38 phosphorylation, the production of nitric oxide (NO), interleukin (IL)-6 and tumor necrosis factor (TNF)-α, the mRNA and protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 were enhanced by lipopolysaccharide (LPS). At concentrations of less than 10 µmol/L, JLU1124 inhibits p38 phosphorylation in a dose-dependent manner and significantly suppresses LPS-induced production of NO, IL-6 and TNF-α, and decreases the expressions of iNOS and COX-2 in RAW264.7 macrophages which indicate that JLU1124 has anti-inflammatory effects. However, JLU1124 has no significant effect on the phosphorylation of extracellular signal-regulated kinase1/2 and c-Jun NH2-terminal kinase which was involved in inflammation. Furthermore, our results showed that JLU1124 inhibits NF-κB inhibitor (IκB)α phosphorylation, nuclear translocation and transcriptional activity of NF-κB induced by LPS which may be through suppression of Akt phosphorylation. In conclusion, our study indicates that JLU1124 efficiently inhibits p38 phosphorylation and has anti-inflammatory effects in LPS-treated RAW264.7 macrophages. The anti-inflammatory mechanism of JLU1124 is mainly through decreasing Akt phosphorylation and inhibiting IκBα phosphorylation, thus suppressing NF-κB activation and nuclear translocation.

The BH3 mimetic S1 induces autophagy through ER stress and disruption of Bcl-2/Beclin 1 interaction in human glioma U251 cells.

Previous results showed that a novel BH3 mimetic S1 could induce cell death in a wide range of cancer types in vitro through Bax/Bak-dependent apoptosis. We demonstrated that in addition to mitochondrial pathway apoptosis, endoplasmic reticulum (ER) stress-associated apoptosis was also induced by S1. Moreover, S1 can induce autophagy in U251 cells, which may occur through ER stress and disruption of the association of Bcl-2 and Beclin 1. Inhibition of autophagy by the autophagic inhibitors 3-methyladenine (3-MA) or chloroquine (CQ) increased S1-induced apoptosis. In conclusion, autophagy plays an important role in S1-induced U251 cell death.