Upregulation of heat shock protein 70 and the differential protein expression induced by tumor necrosis factor-alpha enhances migration and inhibits apoptosis of hepatocellular carcinoma cell HepG2.

Tumor necrosis factor alpha (TNFα) plays diverse roles in liver damage and hepatocarcinogenesis with its multipotent bioactivity. However, the influence of TNFα on protein expression of hepatocellular carcinoma (HCC) is incompletely understood. Therefore, we aimed to investigate the differential protein expression of HCC in response to TNFα stimulus. We observed that HepG2 cell revealed a higher resistance to TNFα-induced apoptosis as compared to the non-tumorigenic hepatocyte THLE-2. By using a label-free quantitative proteomic analysis, we found that 520 proteins were differentially expressed in the HepG2 cells exposed to TNFα, including 211 up-regulated and 309 down-regulated proteins. We further confirmed several proteins with significant expression change (TNFα/control ratio>2.0 or <0.5) by immunoblotting using specific antibodies. We also analyzed the differential expressed proteins using Gene ontology and KEGG annotations, and the results implicated that TNFα might regulate ribosome, spliceosome, antigen processing and presentation, and energy metabolism in HepG2 cells. Moreover, we demonstrated that upregulation of heat shock protein 70 (HSP70) was involved in both the promoted migration and the inhibited apoptosis of HepG2 cells in response to TNFα. Collectively, these findings indicate that TNFα alters protein expression such as HSP70, which triggering specific molecular processes and signaling cascades that promote migration and inhibit apoptosis of HepG2 cells.

Expression of cholecystokinin receptors in colon cancer and the clinical correlation in Taiwan.

Cholecystokinin and gastrin receptors are upregulated in many human digestive malignancies; however, the correlation of their expressions with severity of colon carcinoma remains sketchy. Here, we determined the expression of cholecystokinin-1 and cholecystokinin-2 receptor, CCK1R and CCK2R, in colon carcinomas and investigated their correlations with clinicopathological characteristics and 1-year survival rate. Expression of CCK1R and CCK2R was determined by immunohistochemical assay in tissue samples obtained from 97 surgical specimens. Clinicopathological character analysis revealed that higher expression of cytoplasmic CCK1R and CCK2R was significantly associated with several variables including the depth of tumor invasion (P = 0.001), venous invasion (P = 0.023), and progression stage (P = 0.013). In addition, immunohistochemical staining revealed statistically significant associations of nuclear CCK1R expression with higher lymphatic invasion (P = 0.042), progression stage (P = 0.025), and unfavorable survival (P = 0.025). Interestingly, we found no link between nuclear CCK2R expression and all the clinicopathological characteristics examined. Taken these, our findings indicate that nuclear CCK1R represents a potential biomarker for poor prognosis, and CCK1R may play a role differing from CCK2R in colon carcinogenesis.

AMPK activation inhibits expression of proinflammatory mediators through downregulation of PI3K/p38 MAPK and NF-κB signaling in murine macrophages.

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) plays a central role in energy homeostasis and regulation of inflammatory responses. The present study is aimed to investigate the anti-inflammatory effects of ENERGI-F704, a nucleobase analogue isolated from bamboo leaves, on expression of proinflammatory mediators in murine macrophage RAW264.7 in response to lipopolysaccharide (LPS). ENERGI-F704 enhanced phosphorylation of AMPK(T172) but insignificantly affected the viability of RAW264.7 cells. Further investigation showed that ENERGI-F704 decreased mRNA expression of interleukin (IL)-6, IL-8, tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX2), and inducible nitric oxide synthase (iNOS) induced by LPS, as well as suppressed the production of prostaglandin E2 (PGE2) and nitric oxide (NO). Additionally, the inhibitory effects of ENERGI-F704 on the LPS-induced proinflammatory mediators were diminished by pretreatment of AMPK inhibitor Compound C. ENERGI-F704 also inhibited LPS-triggered activation of nuclear factor kappa B (NF-κB), phosphatidylinositol 3-kinase (PI3K), and p38 mitogen-activated protein kinase (p38), whereas extracellular signal-regulated kinase (Erk)1/2 and c-Jun N-terminal kinase (JNK) were insignificantly influenced. Our findings indicate that ENERGI-F704 may exert anti-inflammatory activity on RAW264.7 cells in response to LPS through the activation of AMPK and suppression of PI3K/P38/NF-κB signaling and the consequent decreased expression of proinflammatory mediators, suggesting that ENERGI-F704 is beneficial to the amelioration of inflammatory disorders.

Anti-inflammatory effects of Perilla frutescens leaf extract on lipopolysaccharide-stimulated RAW264.7 cells.

Perilla leaves are widely used in Chinese herbal medicine and in Japanese herbal agents used to treat respiratory diseases. This study aimed to investigate the anti­inflammatory effects and the underlying mechanisms of Perilla frutescens leaf extract (PLE). Murine macrophage RAW264.7 cells were used as a model. Cell viability and morphological changes were studied by the MTT assay and microscopy. mRNA expression of pro­inflammatory mediators was assessed by both semi­quantitative reverse transcription­polymerase chain reaction (RT­PCR) and quantitative (q) RT­PCR. Nitric oxide (NO) and prostaglandin E2 (PGE2) production were analyzed by the Griess test and sandwich enzyme­linked immunosorbent assay (ELISA), respectively. The activation of kinase cascades was studied by immunoblotting. Our findings showed that PLE slightly affects cell viability, but alleviates LPS­induced activation of RAW264.7 cells. Furthermore, PLE significantly reduced the LPS­induced mRNA expression of the interleukin (IL)­6, IL­8, tumor necrosis factor­α (TNF­α), cyclooxygenase­2 (COX­2) and inducible nitric oxide synthase (iNOS), genes in a dose­dependent manner. In addition, PLE reduced NO production and PGE2 secretion induced by LPS. PLE also inhibited activation of mitogen­activated protein kinases (MAPKs), increased the cytosolic IκBα level, and reduced the level of nuclear factor (NF)­κB. Taken together, these findings indicate that PLE significantly decreases the mRNA expression and protein production of pro­inflammatory mediators, via the inhibition of extracellular­signal­regulated kinase (ERK)1/2, c­Jun N­terminal kinase (JNK), p38, as well as NF­κB signaling in RAW264.7 cells stimulated with LPS.

Inflammatory fibroid polyp of rectum mimicking rectal cancer.

Inflammatory fibroid polyps (IFPs) are rare benign tumors of the rectum. Mutation and activating platelet-derived growth factor receptor alpha (PDGFRA) contribute to tumor development. We present a case of IFPs in the middle rectum that mimic rectal cancer. A 65-year-old woman presented with the symptom of fresh blood in the stool and body weight loss of 6 kg in the preceding 3 weeks. A rectal polypoid tumor was noted upon digital examination. Sigmoidoscopy showed a middle rectal tumor measuring 3 × 2.7 cm with obstruction. Computed tomography (CT) scans of the abdomen showed a rectal tumor that had invaded the sacral bone and was associated with four enlarged lymph nodes greater than 1 cm. The radiological report suggested a diagnosis of rectal cancer with lymph node metastases. To remove the obstruction, the patient was initially treated with excision of the tumor and loop sigmoidal colostomy to the abdomen wall. Total mesorectal resection of rectal and sacral tumor followed 10 days later. Histopathological examination of the rectal and sacral tumor showed proliferation of vessels, fibroblast-like spindle cells, and mixed inflammatory cells, including the plasma cells and eosinophils. The spindle cells were diffusely positive to PDGFRA and were focal positive to CD34 and smooth muscle actin. Based on histopathological and immunohistochemical findings, the diagnosis of IFP is indicated. This was the first reported case of IFPs of the rectum presenting with lymph node enlargement and attachment to the sacrum mimicking rectal cancer.