[Experimental study of the function of nuclear factor-κB-dependent epithelial to mesenchymal transition in pancreatic cancer cells under hypoxic conditions].

OBJECTIVE: To investigate the function of nuclear factor (NF)-κB in the epithelial to mesenchymal transition induced by hypoxia in pancreatic cancer cells. METHODS: For cultured pancreatic cancer cells (BxPC-3 and Panc-1) under hypoxic and normoxic conditions, the differences in the morphology were observed by optical microscope. The expression of markers of epithelial and mesenchymal phenotypes, E-cadherin, vimentin and N-cadherin, were determined by Western blot. NF-κB P65 activity was measured by electrophoretic mobility shift assay. Invasion and gemcitabine resistance of pancreatic cancer cells were evaluated in matrigel invasion assay and cell counting kit-8 assay. Both molecular and pharmacologic means of inhibiting NF-κB P65 were used in these hypoxic cells and then the above resulting phenotypes were compared with those of the control-treated cells. RESULTS: After cultured pancreatic cancer cells under hypoxic conditions for 48 h, normoxic cells exhibited a polygonal shape and formed tight clusters of cells, whereas hypoxic cells took on an elongated, fibroblastoid morphology associated with a more highly invasive character and resistance to gemcitabine; hypoxic cells exhibited an suppression of E-cadherin and increase in vimentin and N-cadherin expression. NF-κB P65 activity was elevated in hypoxic cells. On the contrary, on molecular or pharmacologic inhibition of NF-κB P65, hypoxic cells regained expression of E-cadherin, lost expression of N-cadherin, and reversed their highly invasive and drug resistant phenotype. CONCLUSIONS: Pancreatic cancer cells underwent epithelial to mesenchymal transition exposed to hypoxia, exhibited highly invasive and drug resistant phenotype. Inhibition of NF-κB P65 under hypoxic conditions, pancreatic cancer cells regained expression of E-cadherin, lost expression of N-cadherin, and reversed their highly invasive and drug resistant phenotype.

Immunomodulating Therapy by Picibanil-based Imiquimod and Virotherapy for Advanced Uterine Cancer after Laparoscopic Surgery.

This is a case report of a uterine cancer with the International Federation of Gynecology and Obstetrics staging 3c2 with the initial clinical presentation of postmenopausal vaginal bleeding in August 2015. Endometrium biopsy showed invasive nests of poorly differentiated grade 3 endometrioid adenocarcinoma. The patient received robotic surgery including total hysterectomy, bilateral salpingo-oophorectomy, bilateral pelvic lymph node dissection, para-aortic lymph node dissection, and washing cytology. The final pathology showed an endometrioid carcinoma with myometrium invasion up to 85% and para-aortic and pelvic lymph nodes invasion. The patient received six courses of adjuvant chemotherapy (paclitaxel and carboplatin) with concurrent chemoradiotherapy after the surgery. Later, immunotherapy with Picibanil (OK-432) and interleukin-2 (IL-2) was given, and cancer did not recur for 34 months until tumor recurrence at the liver dome and bilateral lung was noted by positron-emission tomography scan in July 2018. The patient received laparoscopic surgery for intra-abdominal tumor excision in December 2018, and the tumor found extended to the right diaphragm, liver surface, omentum, bilateral flank to pelvic peritoneum, Douglas pouch, and upper rectum. We continued the immunotherapy with OK-432, IL-2, Aldara cream (imiquimod), and later on, virotherapy (human papillomavirus vaccine). The immune risk profiles showed T-cells' proliferation and alteration of the Th1/Th2 activation after immunotherapy and virotherapy. Proctectomy with colon-anal anastomosis and cytoreduction surgery with hyperthermic intraperitoneal chemotherapy (HIPEC) (doxorubicin and paclitaxel) was performed in January 2019. After the surgery, the patient received chemotherapy (topotecan, paclitaxel, lipodox, and carboplatin) and continued the immunotherapy. The immune risk profiles showed CD4, CD4/CD8 increase after HIPEC and immunotherapy. The patient continued the therapy until May 2020.

Protective Effects of Hydrogen Gas on Experimental Acute Pancreatitis.

Acute pancreatitis (AP) is an inflammatory disease mediated by damage to acinar cells and pancreatic inflammation. In patients with AP, subsequent systemic inflammatory responses and multiple organs dysfunction commonly occur. Interactions between cytokines and oxidative stress greatly contribute to the amplification of uncontrolled inflammatory responses. Molecular hydrogen (H2) is a potent free radical scavenger that not only ameliorates oxidative stress but also lowers cytokine levels. The aim of the present study was to investigate the protective effects of H2 gas on AP both in vitro and in vivo. For the in vitro assessment, AR42J cells were treated with cerulein and then incubated in H2-rich or normal medium for 24 h, and for the in vivo experiment, AP was induced through a retrograde infusion of 5% sodium taurocholate into the pancreatobiliary duct (0.1 mL/100 g body weight). Wistar rats were treated with inhaled air or 2% H2 gas and sacrificed 12 h following the induction of pancreatitis. Specimens were collected and processed to measure the amylase and lipase activity levels; the myeloperoxidase activity and production levels; the cytokine mRNA expression levels; the 8-hydroxydeoxyguanosine, malondialdehyde, and glutathione levels; and the cell survival rate. Histological examinations and immunohistochemical analyses were then conducted. The results revealed significant reductions in inflammation and oxidative stress both in vitro and in vivo. Furthermore, the beneficial effects of H2 gas were associated with reductions in AR42J cell and pancreatic tissue damage. In conclusion, our results suggest that H2 gas is capable of ameliorating damage to the pancreas and AR42J cells and that H2 exerts protective effects both in vitro and in vivo on subjects with AP. Thus, the results obtained indicate that this gas may represent a novel therapy agent in the management of AP.

Effects of the HIF-1α and NF-κB loop on epithelial-mesenchymal transition and chemoresistance induced by hypoxia in pancreatic cancer cells.

Hypoxia is a microenvironmental factor which plays a critical role in tumor development and chemoresistance. Epithelial-to-mesenchymal transition (EMT) induced by hypoxia is one of the critical causes of treatment failure and chemoresistance in different types of human cancers. Stabilization of the hypoxia-inducible factor-1α (HIF-1α) transcription complex, caused by intratumoral hypoxia, promotes tumor progression and chemoresistance. Previous evidence suggests that hypoxia can also activate nuclear factor-κB (NF-κB), a known mediator of EMT, which is accompanied by reduced expression of epithelial marker E-cadherin and enhanced expression of the mesenchymal markers Vimentin and N-cadherin as well as overexpression of various transcription factors of EMT, such as Snail and Twist. Based on this evidence, the present study aimed to investigate whether downregulation of the p65 subunit of NF-κB or HIF-1α by small interfering RNA (siRNA) may reverse the EMT phenotype and inhibit the proliferation and induce the apoptosis of pancreatic cancer cell lines (PANC-1, BxPC3) under hypoxic conditions in vitro and enhance the efficacy of gemcitabine in the treatment of pancreatic cancer. These results provide molecular evidence showing that the activation of the HIF-1α and NF-κB loop is mechanistically linked with the chemoresistance phenotype (EMT phenotype) of pancreatic cancer cells under hypoxic conditions, suggesting that the inactivation of HIF-1α and NF-κB signaling by novel strategies may be a potential targeted therapeutic approach for overcoming EMT and chemoresistance induced by hypoxia.

Dihydroartemisinin enhances Apo2L/TRAIL-mediated apoptosis in pancreatic cancer cells via ROS-mediated up-regulation of death receptor 5.

BACKGROUND: Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, has recently shown antitumor activity in various cancer cells. Apo2 ligand or tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) is regarded as a promising anticancer agent, but chemoresistance affects its efficacy as a treatment strategy. Apoptosis induced by the combination of DHA and Apo2L/TRAIL has not been well documented, and the mechanisms involved remain unclear. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report that DHA enhances the efficacy of Apo2L/TRAIL for the treatment of pancreatic cancer. We found that combined therapy using DHA and Apo2L/TRAIL significantly enhanced apoptosis in BxPC-3 and PANC-1 cells compared with single-agent treatment in vitro. The effect of DHA was mediated through the generation of reactive oxygen species, the induction of death receptor 5 (DR5) and the modulation of apoptosis-related proteins. However, N-acetyl cysteine significantly reduced the enhanced apoptosis observed with the combination of DHA and Apo2L/TRAIL. In addition, knockdown of DR5 by small interfering RNA also significantly reduced the amount of apoptosis induced by DHA and Apo2L/TRAIL. CONCLUSIONS/SIGNIFICANCE: These results suggest that DHA enhances Apo2L/TRAIL-mediated apoptosis in human pancreatic cancer cells through reactive oxygen species-mediated up-regulation of DR5.

Dihydroartemisinin inhibits angiogenesis in pancreatic cancer by targeting the NF-κB pathway.

PURPOSE: Dihydroartemisinin (DHA) has recently shown antitumor activity in human pancreatic cancer cells. However, its effect on antiangiogenic activity in pancreatic cancer is unknown, and the mechanism is unclear. This study was aimed to investigate whether DHA would inhibit angiogenesis in human pancreatic cancer. METHODS: Cell viability and proliferation, tube formation of human umbilical vein endothelial cells (HUVECs), nuclear factor (NF)-κB DNA-binding activity, expressions of vascular endothelial growth factor (VEGF), interleukin (IL)-8, cyclooxygenase (COX)-2, and matrix metalloproteinase (MMP)-9 were examined in vitro. The effect of DHA on antiangiogenic activity in pancreatic cancer was also assessed using BxPC-3 xenografts subcutaneously established in BALB/c nude mice. RESULTS: DHA inhibited cell proliferation and tube formation of HUVECs in a time- and dose-dependent manner and also reduced cell viability in pancreatic cancer cells. DHA significantly inhibited NF-κB DNA-binding activity, so as to tremendously decrease the expression of NF-κB-targeted proangiogenic gene products: VEGF, IL-8, COX-2, and MMP-9 in vitro. In vivo studies, DHA remarkably reduced tumor volume, decreased microvessel density, and down-regulated the expression of NF-κB-related proangiogenic gene products. CONCLUSIONS: Inhibition of NF-κB activation is one of the mechanisms that DHA inhibits angiogenesis in human pancreatic cancer. We also suggest that DHA could be developed as a novel agent against pancreatic cancer.

Nuclear factor-κB-dependent epithelial to mesenchymal transition induced by HIF-1α activation in pancreatic cancer cells under hypoxic conditions.

BACKGROUND: Epithelial to mesenchymal transition (EMT) induced by hypoxia is one of the critical causes of treatment failure in different types of human cancers. NF-κB is closely involved in the progression of EMT. Compared with HIF-1α, the correlation between NF-κB and EMT during hypoxia has been less studied, and although the phenomenon was observed in the past, the molecular mechanisms involved remained unclear. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report that hypoxia or overexpression of hypoxia-inducible factor-1α (HIF-1α) promotes EMT in pancreatic cancer cells. On molecular or pharmacologic inhibition of NF-κB, hypoxic cells regained expression of E-cadherin, lost expression of N-cadherin, and attenuated their highly invasive and drug-resistant phenotype. Introducing a pcDNA3.0/HIF-1α into pancreatic cancer cells under normoxic conditions heightened NF-κB activity, phenocopying EMT effects produced by hypoxia. Conversely, inhibiting the heightened NF-κB activity in this setting attenuated the EMT phenotype. CONCLUSIONS/SIGNIFICANCE: These results suggest that hypoxia or overexpression of HIF-1α induces the EMT that is largely dependent on NF-κB in pancreatic cancer cells.