Cyy260, a novel small molecule inhibitor,suppresses non-small cell lung cancer cell growth via JAK2/STAT3 pathway

Non-small cell lung cancer (NSCLC) is a malignant tumor that accounts for the most new cancer cases and cancer-related deaths worldwide, and the proliferation and metastasis of NSCLC are the main reasons for treatment failure and patient death. Traditional chemotherapeutic drugs have low selectivity, which can kill cancer cells and cause damage to normal cells at the same time. Therefore, it is particularly important to study therapies that target cancer cells and to find low-toxicity, high-efficiency anticancer drugs. Cyy260 is a novel small molecule inhibitor that we synthesized for the first time. Here, we investigated the in vitro and in vivo antitumor activities of Cyy260 and explored the underlying mechanisms in NSCLC. Cyy260 had a concentration- and time-dependent inhibitory effect on NSCLC cells, but it was less toxic to normal cells. Cyy260 regulated apoptosis through intracellular and extracellular apoptotic pathways. In addition, Cyy260 could also induce cell cycle arrest, thereby inhibiting cell proliferation. Further analysis of molecular mechanisms showed that the JAK2/STAT3 signaling pathway was involved in the antitumor effect mediated by Cyy260. Analysis of subcutaneously transplanted tumors in mice showed that Cyy260 suppressed tumor growth in vivo. Our results proved that Cyy260 is a novel inhibitor of the JAK2/STAT3 pathway thus may have potential in therapy of NSCLC and other cancers.     

OPB-31121, a novel small molecular inhibitor,disrupts the JAK2/STAT3 pathway and exhibits an antitumor activity in gastric cancer cells

We investigated the mechanisms of action and antitumor effects of OPB-31121, a novel STAT3 inhibitor, in gastric cancer cells. OPB-31121 downregulated JAK2 and gp130 expression and inhibited JAK2 phosphorylation which leads to inhibition of STAT3 phosphorylation. OPB-31121 inhibited constitutively activated and IL-6-induced JAK/STAT signaling pathway. OPB-31121 decreased cell proliferation in both gastric cancer cells and in a xenograft model, induced the apoptosis of gastric cancer cells, inhibited the expression of antiapoptotic proteins, and showed synergism with 5-fluorouracil and cisplatin. Taken together, our study suggests that STAT3 inhibition with OPB-31121 can be tested in patients with gastric cancer.     

Induction of cell-cycle arrest and apoptosis by a novel retinobenzoic-acid derivative, TAC-101, in human pancreatic-cancer cells

In this study, we investigated the effect of a novel retinobenzoic acid, 4-[3,5-bis (trimethylsilyl) benzamido] benzoic acid (TAC-101), on the growth of 4 human pancreatic-cancer cell lines; BxPC-3, MIAPaCa-2, CFPAC-1 and AsPC-1. TAC-101 significantly inhibited the proliferation of BxPC-3 and MIAPaCa-2 cells in a time- and concentration-dependent manner, but not the proliferation of AsPC-1 cells. Furthermore, the anti-proliferative effects of TAC-101 on BxPC-3 and MIAPaCa-2 cells were stronger than those of all-trans retinoic acid. Flow-cytometric analyses indicated that treatment of BxPC-3 with TAC-101 strongly induces cell-cycle arrest at the G1 phase. The cell-cycle arrest induced by TAC-101 was accompanied by reduction of retinoblastoma-gene product (RB) phosphorylation and an increase of 2 cyclin-dependent kinase (CDK) inhibitors, p21(WAF1/Cip1) (p21) and p27Kip1 (p27). TAC-101 also caused a decrease in cyclin A and thymidylate synthase, which are E2F-regulated gene products. No changes were observed in the expression of cyclin D1, cyclin E on CDK2. In addition, Hoechst staining, gel electrophoresis and flow-cytometric analysis indicated that a marked reduction in the number of BxPC-3 cells with TAC-101 was related to the induction of apoptosis. Our results suggest that TAC-101 inhibits the growth of certain pancreatic-cancer cells by means of G1-phase cell-cycle arrest resulting from the reduction of RB phosphorylation and the up-regulation of p21 and p27 as well as the induction of apoptosis. TAC-101 may therefore be a useful agent for new therapeutic strategies focusing on inhibition of pancreatic-cancer-cell proliferation.     

JAK2/STAT3 targeted therapy suppresses tumor invasion via disruption of the EGFRvIII/JAK2/STAT3 axis and associated focal adhesion in EGFRvIII-expressing glioblastoma

Background

As a commonly mutated form of the epidermal growth factor receptor, EGFRvIII strongly promotes glioblastoma (GBM) tumor invasion and progression, but the mechanisms underlying this promotion are not fully understood.

Methods

Through gene manipulation, we established EGFRvIII-, wild-type EGFR-, and vector-expressing GBM cells. We used cDNA microarrays, bioinformatics analysis, target-blocking migration and invasion assays, Western blotting, and an orthotopic U87MG GBM model to examine the phenotypic shifts and treatment effects of EGFRvIII expression in vitro and in vivo. Confocal imaging, co-immunoprecipitation, and siRNA assays detected the focal adhesion-associated complex and their relationships to the EGFRvIII/JAK2/STAT3 axis in GBM cells.

Results

The activation of JAK2/STAT3 signaling is vital for promoting migration and invasion in EGFRvIII-GBM cells. AG490 or WP1066, the JAK2/STAT3 inhibitors, specifically destroyed EGFRvIII/JAK2/STAT3-related focal adhesions and depleted the activation of EGFR/Akt/FAK and JAK2/STAT3 signaling, thereby abolishing the ability of EGFRvIII-expressing GBM cells to migrate and invade. Furthermore, the RNAi silencing of JAK2 in EGFRvIII-expressing GBM cells significantly attenuated their ability to migrate and invade; however, as a result of a potential EGFRvIII-JAK2-STAT3 activation loop, neither EGFR nor STAT3 knockdown yielded the same effects. Moreover, AG490 or JAK2 gene knockdown greatly suppressed tumor invasion and progression in the U87MG-EGFRvIII orthotopic models.

Conclusion

Taken together, our data demonstrate that JAK2/STAT3 signaling is essential for EGFRvIII-driven migration and invasion by promoting focal adhesion and stabilizing the EGFRvIII/JAK2/STAT3 axis. Targeting JAK2/STAT3 therapy, such as AG490, may have potential clinical implications for the tailored treatment of GBM patients bearing EGFRvIII-positive tumors.     

IL-17通过JAK2/STAT3信号通路诱导PD-L1在肝内胆管癌细胞中的表达

目的:探讨白细胞介素-17(interleukin-17,IL-17)对人肝内胆管癌细胞程序性细胞死亡蛋白配体1(programmed cell death ligand 1,PD-L1)表达水平的影响及其潜在机制。方法:使用不同浓度(0 ng/mL、5 ng/mL、10 ng/mL、20 ng/mL、50 ng/mL和100 ng/mL)的IL-17作用于人肝内胆管癌细胞系RBE和HCCC9810,Western blot检测PD-L1的表达情况,并筛选出IL-17的最佳作用浓度。使用最佳作用浓度处理RBE和HCCC9810细胞,收集mRNA、蛋白质。实时定量逆转录-聚合酶链反应（qRT-PCR）检测PD-L1在mRNA水平的表达情况。Western blot检测JAK2/STAT3信号通路的活化情况,并使用AG490阻断信号通路,Western blot检测通路被阻断后,IL-17对RBE及HCCC9810细胞PD-L1表达水平的影响。结果:IL-17诱导了PD-L1在RBE和HCCC9810细胞中的表达,并且PD-L1的上调幅度与IL-17的浓度相关,当浓度为50 ng/mL和100 ng/mL时,对PD-L1的诱导作用最为显著。与对照组相比,50 ng/mL的IL-17可显著刺激PD-L1在mRNA水平的表达(P＜0.01)。同对照组相比,50 ng/mL的IL-17促进了STAT3和JAK2蛋白的磷酸化水平(P＜0.01),但并不影响STAT3和JAK2总蛋白的表达水平(P＞0.05)。在使用AG490阻断JAK2/STAT3信号通路后,IL-17诱导PD-L1蛋白表达的能力明显减弱(P＜0.05)。结论:IL-17可诱导PD-L1在人肝内胆管癌细胞系RBE和HCCC9810中的表达,其机制可能与促进JAK2/STAT3信号通路蛋白的磷酸化水平有关。     

The mTORC1/mTORC2 inhibitor AZD2014 enhances the radiosensitivity of glioblastoma stem-like cells

Background

The mammalian target of rapamycin (mTOR) has been suggested as a target for radiosensitization. Given that radiotherapy is a primary treatment modality for glioblastoma (GBM) and that mTOR is often dysregulated in GBM, the goal of this study was to determine the effects of AZD2014, a dual mTORC1/2 inhibitor, on the radiosensitivity of GBM stem-like cells (GSCs).

Methods

mTORC1 and mTORC2 activities were defined by immunoblot analysis. The effects of this mTOR inhibitor on the in vitro radiosensitivity of GSCs were determined using a clonogenic assay. DNA double strand breaks were evaluated according to γH2AX foci. Orthotopic xenografts initiated from GSCs were used to define the in vivo response to AZD2014 and radiation.

Results

Exposure of GSCs to AZD2014 resulted in the inhibition of mTORC1 and 2 activities. Based on clonogenic survival analysis, addition of AZD2014 to culture media 1 hour before irradiation enhanced the radiosensitivity of CD133+ and CD15+ GSC cell lines. Whereas AZD2014 treatment had no effect on the initial level of γH2AX foci, the dispersal of radiation-induced γH2AX foci was significantly delayed. Finally, the combination of AZD2014 and radiation delivered to mice bearing GSC-initiated orthotopic xenografts significantly prolonged survival as compared with the individual treatments.

Conclusions

These data indicate that AZD2014 enhances the radiosensitivity of GSCs both in vitro and under orthotopic in vivo conditions and suggest that this effect involves an inhibition of DNA repair. Moreover, these results suggest that this dual mTORC1/2 inhibitor may be a radiosensitizer applicable to GBM therapy.     

Induction of apoptosis and down-regulation of Bcl-XL in cancer cells by a novel small molecule, 2[[3-(2,3-dichlorophenoxy)propyl]amino]ethanol

In a search for new anticancer agents, we identified that 2[[3-(2,3-dichlorophenoxy) propyl]amino]ethanol (2,3-DCPE) induced apoptosis more effectively in various cancer cells than in normal human fibroblasts. We further evaluated the cell-killing effects of this compound in vitro in several human cancer cell lines and normal human fibroblasts. A cell viability assay showed that IC(50)s for human colon cancer cell lines LoVo and DLD-1, for human lung cancer cell lines H1299 and A549, and for normal human fibroblasts were 0.89, 1.95, 2.24, 2.69, and 12.6 micro M, respectively. Subsequent studies revealed that 2,3-DCPE could cause cleavage of caspase-8, caspase-3, caspase-9, and poly(ADP-ribose) polymerase and release of cytochrome c in cancer cells but not in normal human fibroblasts. Our data also showed that 2,3-DCPE attenuated the protein level of Bcl-XL and that apoptosis induction by 2,3-DCPE could be blocked by enforced overexpression of Bcl-XL. Our results suggest that 2,3-DCPE might be a potential new anticancer agent.     

Inhibition of AKT survival pathway by a small molecule inhibitor in human endometrial cancer cells

The PTEN (phosphatase and tensin homolog deleted on chromosome 10) tumour suppressor is mutated in 40-50% of human endometrial cancers. PTEN exerts its effects in part via inhibition of the antiapoptotic protein AKT. We demonstrate that two endometrial cancer cell lines that harbour PTEN mutations, Ishikawa and RL95-2, have high levels of phosphorylated AKT and high AKT kinase activity. Two additional endometrial cancer cell lines that express wild-type PTEN, Hec1A and KLE, have little phosphorylated AKT and minimal demonstrable AKT kinase activity. We tested a potential inhibitor of the AKT pathway, API-59CJ-OMe, in these four cell lines. We found that API-59CJ-OMe inhibits AKT kinase activity and induces apoptosis in the Ishikawa and RL95-2 cell lines with high AKT activity, but has little effect on Hec1A and KLE cells without AKT activity. API-59CJ-OMe may therefore have therapeutic potential for those endometrial cancers that harbour PTEN mutations and AKT activation.     

微小RNA-375对鼻咽癌细胞侵袭迁移及JAK2／STAT3 信号通路的影响

目的 探讨微小RNA-375（miR-375）调节鼻咽癌细胞侵袭、迁移的作用及对Janus激酶2／信号转导与转录激活子3（JAK2／STAT3）信号通路的靶向调控。方法 选取鼻咽癌CNE-1细胞并采用Lipofectamine 2000脂质体分别转染miR-375模拟物（miR-375组）和miR-375 阴性对照（NC组）,同时设不行转染的CNE-1细胞为对照组,采用实时荧光定量 PCR（QPCR）检测转染48 h各组miR-375的表达情况;划痕实验和Transwell 小室实验分别检测各组细胞的迁移能力和侵袭能力变化;Western blotting检测JAK2／STAT3信号通路中JAK2、STAT3蛋白的变化情况。结果 QPCR检测显示,miR-375在miR-375组的表达量为10.74±1.21,高于NC组的1.09±0.14和对照组的1.12±0.17,差异均有统计学意义（P＜0.05）;miR-375组的细胞迁移率为（18.22±1.78）％,低于对照组的（43.67±2.60）％和NC组的（49.70±1.31）％,差异均有统计学意义（P＜0.05）。miR-375组的穿膜细胞数为（45.76±4.34）个,亦低于对照组的（144.98±3.65）个和NC组的（126.23±6.32）个,差异均有统计学意义（P＜0.05）;miR 375组JAK2、STAT3蛋白的表达水平分别为0.31±0.6和0.27±0.05,均低于对照组的0.54±0.05和0.41±0.06以及NC组的0.50±0.06和0.43±0.07（P＜0.05）,NC组和对照组以上指标的差异均无统计学意义（P＞0.05）。结论 上调miR-375表达可抑制CNE-1细胞的侵袭和迁移能力,可能与抑制JAK2／STAT3 信号通路的激活有关。     

HO-3867, a STAT3 inhibitor induces apoptosis by inactivation of STAT3 activity in BRCA1-mutated ovarian cancer cells

BRCA1 plays an important role in DNA damage and repair, homologous recombination, cell-cycle regulation and apoptosis. BRCA-mutated ovarian cancer often presents at an advanced stage, however, tend to have better response to platinum-based chemotherapy as compared with sporadic cases of epithelial ovarian cancer (EOC). In spite of this, most patients will develop a recurrence and eventually succumb to the disease. Preclinical studies are currently investigating natural compounds and their analogs for tumor-directed targets in ovarian cancer. The aim of this study is to investigate whether the STAT3 inhibitor HO-3867, a novel curcumin analog, has a therapeutic effect on BRCA1-mutated ovarian cancer. Our novel agent, HO-3867 and a commercial STAT3 inhibitor, STATTIC, significantly inhibited BRCA-mutated ovarian cancer cells in vitro in a dose- and time-dependent manner. BRCA-mutated ovarian cancer cells treated with HO-3867 exhibited a significant degree of apoptosis with elevated levels of cleaved caspase-3, caspase-7 and PARP. HO-3867 treatment induced more reactive oxygen species (ROS) in BRCA-mutated cells compared with wild-type cells, however, there was no increased ROS when benign ovarian surface epithelial cells were treated with HO-3867. BRCA1-mutated cancer cells had higher expression of Tyrosine-phosphorylated STAT3 (pTyr705) as compared with other STAT proteins. Furthermore, treatment of these cells with HO-3867 resulted in decreased expression of pTyr705 and its downstream targets cyclin D1, Bcl-2 and survivin. In addition, overexpression of STAT3 cDNA provided resistance to HO-3867-induced apoptosis. Our results show that HO-3867, a potent STAT3 inhibitor, may have a role as a biologically targeted agent for BRCA1-mutated cancers either as an adjunct to cytotoxic chemotherapy or as a single agent.     

Mesenchymal stem cells enhance lung cancer initiation through activation of IL-6/JAK2/STAT3 pathway

Background

The role of mesenchymal stem cells (MSCs) and IL-6 in lung cancer has not been well-addressed. We aimed to determine if MSCs can enhance the ability of tumor initiation of lung cancer cells, and link MSCs with activation of the IL-6/JAK2/STAT3 signaling pathway.

Materials and methods

Lung cancer cell lines A549 and CL1-5 were directly or indirectly cocultured with MSCs. Spheres were defined as cell colonies with >50% area showing 3-dimensional structure and blurred cell margins. Cells without and with MSCs were injected into NOD/SCID mice. The percentage of tumor formation was determined. The influence of the IL-6/JAK2/STAT3 signaling pathway in cancer cell sphere formation and tumor growth were investigated.

Results

A very small number of lung cancer cells, when mixed with otherwise non-tumorigenic MSCs, obtained de novo tumorigenicity when injected subcutaneously and allowed to form a tumor xenograft. Secretion of IL-6 from MSCs increased activation of the JAK2/STAT3 pathway in cancer cells, and enhanced sphere formation and tumor initiation. A reduced capacity of tumor formation of A549 and CL1-5 lung cancer cells when IL-6 was inhibited in MSCs or STAT3 was silenced in A549 and CL1-5 admixed with MSCs.

Conclusions

Culture of A549 or CL1-5 lung cancer cells with MSCs increased sphere formation, drug resistance, and overexpression of pluripotency markers through activation of the IL-6/JAK2/STAT3 pathway. MSCs enhanced the capability of A549 and CL1-5 lung cancer cells to form tumors in immunodeficient mice. Blockade of the IL-6/JAK2/STAT3 pathway attenuated the capability of A549 and CL1-5 cells to form tumors.