Smac Mimetic Bypasses Apoptosis Resistance in FADD- or Caspase-8-Deficient Cells by Priming for Tumor Necrosis Factor ��-Induced Necroptosis

Searching for new strategies to bypass apoptosis resistance, we investigated the potential of the Smac mimetic BV6 in Jurkat leukemia cells deficient in key molecules of the death receptor pathway. Here, we demonstrate for the first time that Smac mimetic primes apoptosis-resistant, FADD- or caspase-8-deficient leukemia cells for TNFα-induced necroptosis in a synergistic manner. In contrast to TNFα, Smac mimetic significantly enhances CD95-induced apoptosis in wild-type but not in FADD-deficient cells. Interestingly, Smac mimetic- and TNFα-mediated cell death occurs without characteristic features of apoptosis (i.e., caspase activation, DNA fragmentation) in FADD-deficient cells. By comparison, Smac mimetic and TNFα trigger activation of caspase-8, -9, and -3 and DNA fragmentation in wild-type cells. Consistently, the caspase inhibitor zVAD.fmk fails to block Smac mimetic- and TNFα-triggered cell death in FADD- or caspase-8-deficient cells, while it confers protection in wild-type cells. By comparison, necrostatin-1, an RIP1 kinase inhibitor, abolishes Smac mimetic- and TNFα-induced cell death in FADD- or caspase-8-deficient. Thus, Smac mimetic enhances TNFα-induced cell death in leukemia cells via two distinct pathways in a context-dependent manner: it primes apoptosis-resistant cells lacking FADD or caspase-8 to TNFα-induced, RIP1-dependent and caspase-independent necroptosis, whereas it sensitizes apoptosis-proficient cells to TNFα-mediated, caspase-dependent apoptosis. These findings have important implications for the therapeutic exploitation of necroptosis as an alternative cell death program to overcome apoptosis resistance.     

HIF-1α siRNA Leads to Apoptosis of Pancreatic Cancer Cells under Hypoxic Conditions

OBJECTIVE To explore the role of hypoxic inducible factor-1α(HIF-1α) in the proliferation and apoptosis of pancreatic cancer cells under hypoxic conditions.METHODS A cassette encoding small interference RNA (siRNA)targeting HIF-1α mediated by recombinant adeno-associated virus (rAAV) was constructed, giving rAAV-siHIF, rAAV-siHIF or rAAV- hrGFP was transfected into exponentially growing MiaPaCa2 cells under hypoxic conditions. Then, the expression of HIF-1αmRNA and protein, the proliferation and apoptosis of MiaPaCa2 cells were examined, using real-time PCR, Western Blot, MTT and TUNEL, respectively.RESULTS Under hypoxic conditions, rAAV-siHIF inhibited the expression of HIF-1α mRNA and protein in MiaPaCa2 cells. At the same time, rAAV-siHIF decreased MiaPaCa2 cell proliferation and induced apoptosis. However, rAAV-hrGFP had no effect on the expression of HIF-1α as well as the proliferation and apoptosis of MiaPaCa2 cells under hypoxic conditions.CONCLUSION Under hypoxic conditions, HIF-1α plays a key role in the proliferation of MiaPaCa2 cells, and inhibition of HIF-1α expression can lead to MiaPaCa2 cell apoptosis.     

Effect of Celecoxib on Apoptosis of Endometrial Carcinoma Cell

Objective:To investigate the effect of Celecoxib on proliferation and apoptosis of the endometrial carcinoma cell HEC-1B and the effect on the expression of Fas and Survivin mRNA.Methods:The inhibition on the growth of human endometrial carcinoma cell HEC-1B was investigated by cell culture and MTT experiment when treated with different concentrations of Celecoxib.The cell apoptosis was detected by flow cytometry and DNA Ladder Electrophoresis.The change of the expression of Fas and Survivin mRNA after the treatment of Celecoxib was detected With RT-PCR.Results:Celecoxib could effectively inhibit the growth of HEC-1B cells and induce apoptosis.Survivin mRNA expression was decreased and Fas mRNA expression was increased after treating with Celecoxib.Conclusion:Celecoxib could inhibit HEC-1B cell proliferation and induce its apoptosis.     

Stromal Cell-Derived Factor 1�� Mediates Resistance to mTOR-Directed Therapy in Pancreatic Cancer

Purpose

The factors preventing the translation of preclinical findings supporting the clinical development mTOR-targeted therapy in pancreatic cancer therapy remain undetermined. Stromal cell.derived factor 1α (SDF-1α)-CXCR4 signaling was examined as a representative microenvironmental factor able to promote mTOR-targeted therapy resistance in pancreatic cancer.

Experimental Design

Primary pancreas explant xenografts and in vitro experiments were used to perform pharmacodynamic analyses of SDF-1α-CXCR4 regulation of the mTOR pathway. Combinatorial effects of CXCR4, EGFR, and mTOR pharmacologic inhibition were evaluated in temsirolimus-resistant and -sensitive xenografts. Intratumoral gene and protein expressions of mTOR pathway effectors cyclin D1, c-Myc, and VEGF were evaluated.

Results

Baseline intratumoral SDF-1α gene expression correlated with temsirolimus resistance in explant models. SDF-1α stimulation of pancreatic cells resulted in CXCR4-mediated PI3-kinase-dependent S6-RP phosphorylation (pS6-RP) on exposure to temsirolimus. Combinatorial therapy with AMD3465 (CXCR4 small-molecule inhibitor) and temsirolimus resulted in effective tumor growth inhibition to overcome temsirolimus resistance. In contrast, SDF-1α exposure induced a temsirolimus-resistant phenotype in temsirolimus-sensitive explants. AMD3465 inhibited CXCR4-mediated intratumoral S6-RP phosphorylation and cyclin D and c-myc gene expression. Next, CXCR4 promoted intratumoral EGFR expression in association with temsirolimus resistance. Treatment with AMD3465, temsirolimus- and erlotinib-mediated tumor growth inhibition to overcome temsirolimus resistance in the explant model. Lastly, SDF-1α-CXCR4 signaling increased intratumoral VEGF gene and protein expression.

Conclusions

SDF-1α-CXCR4 signaling represents a microenvironmental factor that can maintain mTOR pathway fidelity to promote resistance to mTOR-targeted therapy in pancreatic cancer by a variety of mechanisms such as recruitment of EGFR signaling and angiogenesis.     

Attenuation of Telomerase Activity by siRNA Targeted Telomerase RNA Leads to Apoptosis and Inhibition of Proliferation in Human Renal Carcinoma Cells

OBJECTIVE Telomerase is an attractive molecular target for cancer therapy because the activation of telomerase is one of the key steps in cell immortalization and carcinogenesis. RNA interference using small-interfering RNA (siRNA) has been demonstrated to be an effective method for inhibiting the expression of a given gene in human cells. The aim of the present study was to investigate whether inhibition of telomerase activity by siRNA targeted against human telomerase RNA (hTR) can inhibit proliferation and induce apoptotic cell death in human renal carcinoma cells (HRCCs). METHODS The siRNA duplexes for hTR were synthesized and 786-0 HRCCs were transfected with different concentrations of hTR-siRNA. The influence on the hTR mRNA level, telomerase activity, as well as the effect on cell proliferation and apoptosis was examined. RESULTS Anti-hTR siRNA treatment of HRCCs resulted in specific reduction of hTR mRNA and inhibition of telomerase activity. Additionally, significant inhibition of proliferation and induction of apoptosis were observed. CONCLUSION siRNA against the hTR gene can inhibit proliferation and induce apoptosis by blocking telomerase activity of HRCCs. Specific hTR inhibition by siRNA represents a promising new option for renal cancer treatment.     

Effects of TNF Secreting HEK Cells on B Lymphocytes’ Apoptosis in Human Chronic Lymphocytic Leukemias

Background: Tumor necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL) is an antitumorcandidate in cancer therapy. This study focused on effects of TRAIL, as a proapototic ligand that causes apoptosis,in B-CELL chronic lymphocytic leukemia cells (B-CLL) . Materials and Methods: A population of HEK 293cells was transducted by lentivirus that these achieved ability for producing the TRAIL protein and then HEK293 cells transducted were placed in the vicinity of CLL cells. After 24 hours of co-culture, apoptosis of CLLcells was assessed by annexin V staining. Results: The amount of Apoptosis was examined separately in fourgroups: 293 HEK TRAIL (16.17±1.04%); 293 HEK GFP (2.7±0.57%); WT 293 HEK (2±2.6%); and CLL cells(0.01±0.01%). Among the groups studied, the maximum amount of apoptosis was in the group that the vectorencoding TRAIL was transducted. In this group, the mean level of soluble TRAIL in the culture medium was253pg/ml; also flow cytometry analyzes showed that proapotosis in this group was 32.8±1.6%, which was higherthan the other groups. Conclusions: In this study, we have demonstrated that TNF secreted from HEK 293 cellsare effective in death of CLL cells.     

I��B Kinases Modulate the Activity of the Androgen Receptor in Prostate Carcinoma Cell Lines

Enhanced nuclear localization of nuclear factor κB (NF-κB) in prostate cancer (PCa) samples and constitutive NF-κB signaling in a class of PCa cell lines with low androgen receptor (AR) expression (PC3 and DU-145) imply an important role of the IκB kinase (IKK)/NF-κB system in PCa. However, most PCa and PCa cell lines depend on the activity of the AR, and the role of NF-κB in these AR-expressing PCa remains unclear. Here, we demonstrate that inhibition of NF-κB signaling by the IKK inhibitor BMS345541 reduced proliferation and increased apoptosis in AR-expressing PCa cell lines. Furthermore, AR activity and target gene expression were distinctively reduced, whereas AR protein levels remained unaltered on BMS345541 treatment. Similar effects were observed particularly after small interfering RNA (siRNA)-mediated knockdown of IKK1, but not by siRNA-mediated suppression of IKK2. Moreover, IKK1 overexpression augmented 5α-dihydrotestosterone-induced nuclear AR translocation, whereas nuclear AR was reduced by IKK1 knockdown or BMS345541. However, because IKK1 also enhances the activity of a chronically nuclear AR mutant, modulation of the subcellular distribution seems not to be the only mechanism by which IKK1 enhances AR activity. Finally, reduced in vivo AR phosphorylation after BMS345541 treatment and in vitro AR phosphorylation by IKK1 or IKK2 imply that AR constitutes a novel IKK target. Taken together, our data identify IKK1 as a potentially target structure for future therapeutic intervention in PCa.     

Fentanyl Increases Colorectal Carcinoma Cell Apoptosis by Inhibition of NF-κB in a Sirt1-dependent Manner

Background: Fentanyl is used as an analgesic to treat pain in a variety of patients with cancer and recentlyit has become considered to also act as an antitumor agent. The study present was designed to investigate theeffects of fentanyl on colorectal cancer cell growth and plausible mechanisms. Materials and Methods: The humancolorectal carcinoma cell line HCT116 was subcutaneously injected into nude mice. The viability of HCT116 wastested by MTT assay, and apoptosis by flow cytometry and caspase-3 activity. The expression of Sirt1 and NF-κB were evaluated by Western blotting and the levels of Sirt1 and NF-κB by fluorescence method. SiRNA wasused to silence and Ad-Sirt1 to overexpress Sirt1. Results: Our data showed that fentanyl could inhibit tumorgrowth, with increased expression of Sirt1 and down-regulation of Ac-p65 in tumors. Compared with controlcells without treatment, HCT116 cells that were incubated with fentanyl had a higher apoptotic rate. Moreover,fentanyl could increase expression and activity of Sirt1 and inhibitor expression and activity of NF-κB, whichmight be mechanisms of fentanyl action. Conclusions: Fentanyl increased colorectal carcinoma cell apoptosisby inhibition of NF-κB activation in a Sirt1-dependent manner.     

Evaluation of Intraoperative Radiotherapy for Gastric Carcinoma

OBJECTIVE To study the proper sites and doses of intraoperative radiotherapy (IORT) for gastric carcinoma and the effects of this treatment.METHODS A total of 106 cases of stage Ⅰ- Ⅳ gastric carcinoma who received a D2 or D3 radical resection operation combined with IORT were analyzed. Sixty-seven patients with gastric cancer of the antrum and body received distal gastrectomy. The sites of irradiation were at the celiac artery and hepatoduodenal ligament area. Another 39 patients with carcinoma of the cardia and upper part of the gastric body and whole stomach received proximal gastrectomy or total gastrectomy. The sites of irradiation for this group were the upper margin of the pancreas and the regional paraaorta.The therapeutic effects (including survival and complications) of these 106cases who received a combined operation IORT (IORT group) were compared with 441 cases treated during the same time period by a radical resection operation alone (operation group).RESULTS The radiation dose below 30 Gy was safe. The therapeutic method of the operation combined with IORT did not prolong the survival time of patients with stage Ⅱ and Ⅳ gastric cancer, but the 5-year survival rates of patients with stage Ⅱ and Ⅲ gastric cancers were significantly improved.While the 5-year survival rates of the stage Ⅲ cancer patients receiving D2 resection combined with IORT had marked improvement, for those receiving a D3 radical resection, only the postoperative survival rates at 3 and 4 years of those cases in stages Ⅲ cancers were improved (P＜0.005-0.001). The 5-year survival rate for those patients was raised only 4.7%(P＞0.05).CONCLUSION The 5-year survival rates of patients with stages Ⅱ and Ⅲ gastric carcinoma who received a D2 lymphadenectomy combined with IORT were improved and had no influence on the postoperative complications and mortality.     

A Novel Strategy to Co-target Estrogen Receptor and Nuclear Factor κB Pathways with Hybrid Drugs for Breast Cancer Therapy

Nearly 75% of breast tumors express estrogen receptor (ER), and will be treated with endocrine therapy, such as selective estrogen receptor modulator (SERM), tamoxifen, or aromatase inhibitors. Despite their proven success, as many as 40–50% of ER+ tumors fail to respond to endocrine therapy and eventually recur as aggressive, metastatic cancers. Therefore, preventing and/or overcoming endocrine resistance in ER+ tumors remains a major clinical challenge. Deregulation or activation of the nuclear factor κB (NFκB) pathway has been implicated in endocrine resistance and poor patient outcome in ER+ tumors. As a consequence, one option to improve on existing anti-cancer treatment regimens may be to introduce additional anti-NFκB activity to endocrine therapy drugs. Our approach was to design and test SERM-fumarate co-targeting hybrid drugs capable of simultaneously inhibiting both ER, via the SERM, raloxifene, and the NFκB pathway, via fumarate, in breast cancer cells. We find that the hybrid drugs display improved anti-NFκB pathway inhibition compared to either raloxifene or fumarate. Despite some loss in potency against the ER pathway, these hybrid drugs maintain anti-proliferative activity in ER+ breast cancer cells. Furthermore, these drugs prevent clonogenic growth and mammosphere formation of ER+ breast cancer cells. As a proof-of-principle, the simultaneous inhibition of ER and NFκB via a single bifunctional hybrid drug may represent a viable approach to improve the anti-inflammatory activity and prevent therapy resistance of ER-targeted anti-cancer drugs.     

Neurogenin 3–Directed Cre Deletion of Tsc1 Gene Causes Pancreatic Acinar Carcinoma

The role of tuberous sclerosis complex (TSC) in the pathogenesis of pancreatic cancers remains largely unknown. The present study shows that neurogenin 3 directed Cre deletion of Tsc1 gene induces the development of pancreatic acinar carcinoma. By cross-breeding the Neurog3-cre mice with Tsc1^loxp/loxp mice, we generated the Neurog3-Tsc1−/− transgenic mice in which Tsc1 gene is deleted and mTOR signaling activated in the pancreatic progenitor cells. All Neurog3-Tsc1−/− mice developed notable adenocarcinoma-like lesions in pancreas starting from the age of 100 days old. The tumor lesions are composed of cells with morphological and molecular resemblance to acinar cells. Metastasis of neoplasm to liver and lung was detected in 5% of animals. Inhibition of mTOR signaling by rapamycin significantly attenuated the growth of the neoplasm. Relapse of the neoplasm occurred within 14 days upon cessation of rapamycin treatment. Our studies indicate that activation of mTOR signaling in the pancreatic progenitor cells may trigger the development of acinar carcinoma. Thus, mTOR may serve as a potential target for treatment of pancreatic acinar carcinoma.Abbreviations: ACC, acinar cell carcinoma; 4EBP-1, 4E binding protein 1; mTOR, mammlian target of rapamycin; Neurog3, neurogenin 3; PDA, pancreatic ductal adenocarcinoma; S6, ribosomal protein S6; TSC, tuberous sclerosis complex     

A Pilot Study of Chronochemotherapy for Nasopharyngeal Carcinoma

T he rationale of chronochemotherapy is based on the relationship between the pharmarcokinetics of anti-tumor drugs and the circa- dian rhythm, with the concept that anti-tumor drug administration can be delivered at the time of minimized cytotoxic side effects. If the an- ti-tumor drugs are delivered on an individualized chronochemothera- peutic basis, the efficacy of the response may be better with lower side effects. More and more clinical studies have shown that anti-tumor drugs such as ox…