Ming Yu

Bcl-2 Inhibitors Sensitize Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Induced Apoptosis by Uncoupling of Mitochondrial Respiration in Human Leukemic CEM Cells

Previous studies have shown that the lymphoblastic leukemia CEM cell line is resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis because of a low expression of caspase-8. Bcl-2 inhibitors, BH3I-2′ and HA14-1, are small cell-permeable nonpeptide compounds, are able to induce apoptosis by mediating cytochrome c release, and also lead to dissipation of the mitochondrial membrane potential (ΔΨm). This study aimed to use the Bcl-2 inhibitors to sensitize CEM cells to TRAIL-induced apoptosis by switching on the mitochondrial apoptotic pathway. We found that a low dose of BH3I-2′ or HA14-1, which did not induce cytochrome c release, greatly sensitized CEM cells to TRAIL-induced apoptosis. In a similar manner to the classical uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), both BH3I-2′ and HA14-1 induced a reduction in ΔΨm, a generation of reactive oxygen species (ROS), an increased mitochondrial respiration, and a decreased ATP synthesis. This uncoupling function of the Bcl-2 inhibitors was responsible for the synergy with TRAIL-induced apoptosis. CCCP per se did not induce apoptosis but again sensitized CEM cells to TRAIL-induced apoptosis by uncoupling mitochondrial respiration. The uncoupling effect facilitated TRAIL-induced Bax conformational change and cytochrome c release from mitochondria. Inhibition of caspases failed to block TRAIL-mediated cell death when mitochondrial respiration was uncoupled. We observed that BH3I-2′, HA14-1, or CCCP can overcome resistance to TRAIL-induced apoptosis in TRAIL-resistant cell lines, such as CEM, HL-60, and U937. Our results suggest that the uncoupling of mitochondrial respiration can sensitize leukemic cells to TRAIL-induced apoptosis. However, caspase activation per se does not represent an irreversible point of commitment to TRAIL-induced cell death when mitochondrial respiration is uncoupled.

Leptin upregulates telomerase activity and transcription of human telomerase reverse transcriptase in MCF-7 breast cancer cells

The aim was to analyze the mechanism of leptin-induced activity of telomerase in MCF-7 breast cancer cells. We found that leptin activated telomerase in a dose-dependent manner; leptin upregulated the expression of Human Telomerase Reverse Transcriptase (hTERT) at mRNA and protein levels; blockade of signal transducer and activator of transcription 3 (STAT3) phosphorylation significantly counteracted leptin-induced hTERT transcription and protein expression; chromatin immunoprecipitation analysis showed that leptin enhanced the binding of STAT3 to the hTERT promoter. This study uncovers a new mechanism of the proliferative effect of leptin on breast cancer cells and provides a new explanation of obesity-related breast cancer.

Hypoxia-inducible factor-1α regulates chemotactic migration of pancreatic ductal adenocarcinoma cells through directly transactivating the CX3CR1 gene.

CX3CR1 is an important chemokine receptor and regulates the chemotactic migration of pancreatic ductal adenocarcinoma (PDAC) cells. Up to now, its regulatory mechanism remains largely undefined. Here, we report that hypoxia upregulates the expression of CX3CR1 in pancreatic cancer cells. When hypoxia-inducible factor (HIF)-1α expression was knocked down in vitro and in vivo, the expression of CX3CR1 was significantly decreased. Chromatin immunoprecipitation assay demonstrated that HIF-1α bound to the hypoxia-response element (HRE; 5′-A/GCGTG-3′) of CX3CR1 promoter under normoxia, and this binding was significantly enhanced under hypoxia. Overexpression of HIF-1α significantly upregulated the expression of luciferase reporter gene under the control of the CX3CR1 promoter in pancreatic cancer cells. Importantly, we demonstrated that HIF-1α may regulate cancer cell migration through CX3CR1. The HIF-1α/CX3CR1 pathway might represent a valuable therapeutic target to prevent invasion and distant metastasis in PDAC.

Hypoxia inducible factor (HIF)-1α directly activates leptin receptor (Ob-R) in pancreatic cancer cells.

The aim of this study is to investigate the regulatory mechanism of leptin receptors (Ob-R) in pancreatic cancer. We found that the over-expression of hypoxia inducible factor (HIF-1)α and hypoxia up-regulated the expression of Ob-R in pancreatic cancer cells. When HIF-1α gene was silenced in vitro, the expression of Ob-R was significantly decreased. Xenograft mouse models showed that the inhibition of HIF-1α resulted in the concomitant decrease of Ob-R in vivo. In addition, HIF-1α expression was correlated with Ob-R in pancreatic cancer tissues by immunohistochemical staining. Clinical data showed that over-expression of HIF-1 was associated with pathological tumor node metastasis stage, lymph node metastasis and overall survival. HIF-1α directly bound to the hypoxia-responsive element (HRE) located in Ob-R gene promoter (-828/-832) and activated the transcription. Finally, we demonstrated that the silence of HIF-1α gene reversed the inhibitory effect of leptin/Ob-R in pancreatic cancer cells. Taken together, our results indicate that HIF-1α directly regulated Ob-R expression in pancreatic cancer, which might be a valuable therapeutic target for pancreatic cancer.

Cancer-FOXP3 directly activated CCL5 to recruit FOXP3 + Treg cells in pancreatic ductal adenocarcinoma

Forkheadbox protein 3 (FOXP3), initially identified as a key transcription factor for regulatory T cells (Treg cells), was also expressed in many tumors including pancreatic ductal adenocarcinoma (PDAC). However, its role in PDAC progression remains elusive. In this study, we utilized 120 PDAC tissues after radical resection to detect cancer-FOXP3 and Treg cells by immunohistochemistry and evaluated clinical and pathological features of these patients. Cancer-FOXP3 was positively correlated with Treg cells accumulation in tumor tissues derived from PDAC patients. In addition, high cancer-FOXP3 expression was associated with increased tumor volumes and poor prognosis in PDAC especially combined with high levels of Treg cells. Overexpression of cancer-FOXP3 promoted the tumor growth in immunocompetent syngeneic mice but not in immunocompromised or Treg cell-depleted mice. Furthermore, CCL5 was directly trans-activated by cancer-FOXP3 and promoted the recruitment of Treg cells from peripheral blood to the tumor site in vitro and in vivo. This finding has been further reinforced by the evidence that Treg cells recruitment by cancer-FOXP3 was impaired by neutralization of CCL5, thereby inhibiting the growth of PDAC. In conclusion, cancer-FOXP3 serves as a prognostic biomarker and a crucial determinant of immunosuppressive microenvironment via recruiting Treg cells by directly trans-activating CCL5. Therefore, cancer-FOXP3 could be used to select patients with better response to CCL5/CCR5 blockade immunotherapy.

Four types of fatty acids exert differential impact on pancreatic cancer growth.

Increased fatty acids (FAs) regulate pancreatic cancer progression, however, the detailed mechanism is not clear, and different forms of FAs may play diversified roles in pancreatic cancer. To elucidate the underlying mechanism, we compared the effects of four major types of FAs on pancreatic cancer growth both in cell culture and in a mouse model. HPAF pancreatic cancer cells were implanted in nude mice for 14 weeks, and the mice were fed with four different high-fat/high-energy diets (15% fat, 4 kcal/g), an iso-caloric diet (5% fat, 4 kcal/g) and a normal diet (4% fat, 3 kcal/g). The high fat diets were rich in saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and n-6 and n-3 polyunsaturated fatty acids (n6- and n3PUFAs), respectively. While n3PUFA diet decreased tumor viability, the other high fat diets stimulated tumor viability by apparently different mechanisms. For instance, xenografts whose carriers were fed with SFA diet had marked expression of cancer-related proteins and lipid droplets. Although mice that were fed with MUFA- and n6PUFA diets had pancreatic tumors of similar size, liver metastasis occurred more frequently in those with the n6PUFA diet. In experiments in vitro, the HPAF-cell population was increased by SFAs and MUFAs, decreased by n3PUFAs and not changed by n6PUFAs. In conclusion, different fatty acids have different impact on pancreatic cancer cells. The effects of fatty acids on pancreatic cancer cells were consistent in vivo and in vitro except that n6PUFAs only had regulatory effects in vivo.

Arsenic trioxide plus PX-478 achieves effective treatment in pancreatic ductal adenocarcinoma

Arsenic trioxide (ATO) has been selected as a promising treatment not only in leukemia but also in solid tumors. Previous studies showed that the cytotoxicity of ATO mainly depends on the induction of reactive oxygen species. However, ATO has only achieved a modest effect in pancreatic ductal adenocarcinoma, suggesting that the existing radical scavenging proteins, such as hypoxia inducible factor-1, attenuate the effect. The goal of this study is to investigate the effect of combination treatment of ATO plus PX-478 (hypoxia-inducible factor-1 inhibitor) and its underlying mechanism. Here, we showed that PX-478 robustly strengthened the anti-growth and pro-apoptosis effect of ATO on Panc-1 and BxPC-3 pancreatic cancer cells in vitro. Meanwhile, in vivo mouse xenograft models also showed the synergistic effect of ATO plus PX-478 compared with any single agent. Further studies showed that the anti-tumor effect of ATO plus PX-478 was derived from the reactive oxygen species-induced apoptosis. We next confirmed that Hypoxia-inducible factor-1 cleared reactive oxygen species by its downstream target, forkhead box O transcription factors, and this effect may justify the strategy of ATO plus PX-478 in the treatment of pancreatic cancer.

The CX3CL1/CX3CR1 reprograms glucose metabolism through HIF-1 pathway in pancreatic adenocarcinoma

One of the hallmarks of cancer is revised glucose metabolism that promotes cell survival and proliferation. In pancreatic cancer, the regulatory mechanism of glucose metabolism remains to be elucidated. In this study, we found that CX3CR1 is expressed in pancreatic cancer cells lines. Exogenous or transfected CX3CL1 increased glucose uptake and lactate secretion. CX3CL1 stimulated HIF‐1 expression through PI3K/Akt and MAPK pathways. Furthermore, knockdown of HIF‐1 blocked CX3CL1‐modified glucose metabolism in pancreatic adenocarcinoma cells. In conclusion, the CX3CL1/CX3CR1 reprograms glucose metabolism through HIF‐1 pathway in pancreatic cancer cells. J. Cell. Biochem. 114: 2603–2611, 2013.

The alpha-5 helix of Bax is sensitive to ubiquitin-dependent degradation

The pro-apoptotic protein Bax is instable in many cancer cells but the mechanism of Bax degradation remains unclear. Four different lengths of deductive Bax degradation sensitive (BDS) sequences within BH3-BH1 region, BDS-1 (Bax 67-124), BDS-3 (Bax 74-107), BDS-5 (Bax 67-107), and BDS-7 (Bax 74-124), were tested for the susceptibility to ubiquitin-dependent degradation. Both BDS-1 and BDS-7 which contain the alpha5 helix, a putative pore-forming domain of Bax, are sensitive to proteasome-dependent degradation and ubiquitin-conjugation. The Bax alpha5-deletion mutant (Bax-Deltaalpha5) was stable and also maintained its apoptosis-inducing ability. Deletion of helices alpha1 and part of alpha2 (Bax-Delta1-66) or helices alpha3 and alpha4 (Bax-Deltaalpha3,4) did not affect the sensitivity to degradation. However, Bax-Delta1-66 mutant was not able to induce apoptosis. Thus, we propose that the alpha5 helix of Bax is sensitive to ubiquitin-dependent degradation. Moreover, Bax mutant retains its pro-apoptosis ability when the alpha5 helix is deleted.