Inhibition of constitutively activated Stat3 correlates with altered Bcl-2/Bax expression and induction of apoptosis in mycosis fungoides tumor cells.

The Jak/Stat signaling pathway transmits signals from many cytokine and growth factor receptors to target genes in the nucleus. Constitutive activation of Stat3 has recently been observed in many tumor cells and dysregulation of the Stat signaling pathway has been proposed to be implicated in malignant transformation. In a previous study, we found constitutively tyrosine phosphorylated Stat3 in mycosis fungoides tumor cells. Here, we show that the Jak kinase inhibitor, Ag490, inhibits the constitutive binding of Stat3 to an oligonucleotide representing the Stat-binding sequence from the ICAM promotor. The decreased ability of Stat3 to bind DNA precedes dynamic alterations in the expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins (decreased Bcl-2 expression and increased Bax expression) and induction of apoptosis. Thus, our data suggest that the involvement of Stat3 in oncogenic transformation could be mediated through regulation of survival signals.     

Novel synthetic derivatives of the natural product berbamine inhibit Jak2/Stat3 signaling and induce apoptosis of human melanoma cells

Persistent Jak/Stat3 signal transduction plays a crucial role in tumorigenesis and immune development. Activated Jak/Stat3 signaling has been validated as a promising molecular target for cancer therapeutics discovery and development. Berbamine (BBM), a natural bis-benzylisoquinoline alkaloid, was identified from the traditional Chinese herbal medicine Berberis amurensis used for treatment of cancer patients. While BBM has been shown to have potent antitumor activities with low toxicity in various cancer types, the molecular mechanism of action of BBM remains largely unknown. Here, we determine the antitumor activities of 13 synthetic berbamine derivatives (BBMDs) against human solid tumor cells. BBMD3, which is the most potent in this series of novel BBMDs, exhibits over 6-fold increase in biological activity compared to natural BBM. Moreover, BBMD3, directly inhibits Jak2 autophosphorylation kinase activity in vitro with IC₅₀ 0.69 μM. Autophosphorylation of Jak2 kinase at Tyr1007/1008 sites also was strongly inhibited in the range of 15 μM of BBMD3 in human melanoma cells at 4 h after treatment. Following inhibition of autophosphorylation of Jak2, BBMD3 blocked constitutive activation of downstream Stat3 signaling in melanoma cells. BBMD3 also down-regulated expression of the Stat3 target proteins Mcl-1and Bcl-x_L, associated with induction of apoptosis. In sum, our findings demonstrate that the novel berbamine derivative BBMD3 is an inhibitor of the Jak2/Stat3 signaling pathway, providing evidence for a molecular mechanism whereby BBMD3 exerts at least in part the apoptosis of human melanoma cells. In addition, BBMD3 represents a promising lead compound for development of new therapeutics for cancer treatment.     

NV‐128, a novel isoflavone derivative,induces caspase‐independent cell death through the Akt/mammalian target of rapamycin pathway

BACKGROUND:

Resistance to apoptosis is 1 of the key events that confer chemoresistance and is mediated by the overexpression of antiapoptotic proteins, which inhibit caspase activation. The objective of this study was to evaluate whether the activation of an alternative, caspase‐independent cell death pathway could promote death in chemoresistant ovarian cancer cells. The authors report the characterization of NV‐128 as an inducer of cell death through a caspase‐independent pathway.

METHODS:

Primary cultures of epithelial ovarian cancer (EOC) cells were treated with increasing concentration of NV‐128, and the concentration that caused 50% growth inhibition (GI₅₀) was determined using a proprietary assay. Apoptotic proteins were characterized by Western blot analyses, assays that measured caspase activity, immunohistochemistry, and flow cytometry. Protein‐protein interactions were determined using immunoprecipitation. In vivo activity was measured in a xenograft mice model.

RESULTS:

NV‐128 was able to induce significant cell death in both paclitaxel‐resistant and carboplatin‐resistant EOC cells with a GI₅₀ between 1 μg/mL and 5 μg/mL. Cell death was characterized by chromatin condensation but was caspase‐independent. The activated pathway involved the down‐regulation of phosphorylated AKT, phosphorylated mammalian target of rapamycin (mTOR), and phosphorylated ribosomal p70 S6 kinase, and the mitochondrial translocation of beclin‐1 followed by nuclear translocation of endonuclease G.

CONCLUSIONS:

The authors characterized a novel compound, NV‐128, which inhibits mTOR and promotes caspase‐independent cell death. The current results indicated that inhibition of mTOR may represent a relevant pathway for the induction of cell death in cells resistant to the classic caspase‐dependent apoptosis. These findings demonstrate the possibility of using therapeutic drugs, such as NV‐128, which may have beneficial effects in patients with chemoresistant ovarian cancer. Cancer 2009. © 2009 American Cancer Society.     

AKT activation and response to interferon-beta in human cancer cells

Significant growth inhibition and induction of apoptosis by IFN-beta in cancer cells including colorectal cancer cells have been observed. We and others have previously reported the Stat 1 induction of TRAIL is a crucial step in the IFN-beta induced apoptosis pathway. However, when evaluating the sensitivity of a panel of colorectal cancer cell lines, we found no clear correlation between activation of the Jak/Stat signaling pathway and response to interferon. In the present study, we have evaluated the interaction of the PI3k/Akt pathway and IFN-beta induced apoptosis in human colorectal cancer cells. The results demonstrate a correlation between Akt activity, phosphorylation of Bad and resistance to interferon-induced apoptosis in these cells. The association of activation of Akt, phosphorylation of Bad and resistance to IFN-beta-induced apoptosis was further supported by the observation that disruption of the pathway in a more resistant cell line led to sensitization, and expression of an activated Akt in a more sensitive cell line led to increased resistance. Taken together, this data indicates that the PI3/Akt kinase pathway may be an important contributor to IFN-beta sensitivity and resistance in colorectal cancer cells. This data demonstrates a potential pathway by which cells may develop resistance to IFN, and further elucidation of this process may allow us to better target IFN therapy.     

沉默Stat3对卵巢癌细胞系OV2008恶性表型的影响

探讨沉默信号转导和转录活化因子（Stat3）对卵巢癌细胞系OV2008恶性表型的影响。方法：腺病毒M4感染卵巢癌细胞系OV2008后,利用RT-PCR法检测Stat3 mRNA的表达水平;Western blot法检测Stat3蛋白的表达水平;MTT法检测沉默Stat3对OV2008细胞的增殖抑制的影响;Transwell小室法检测沉默Stat3对OV2008细胞的侵袭运动能力的影响;流式细胞术检测沉默Stat3对OV2008细胞的凋亡的影响。结果：RT-PCR以及Western blot结果显示,腺病毒M4感染卵巢癌细胞系OV2008后,OV2008细胞中的Stat3 mRNA以及Stat3蛋白的表达水平均明显降低;MTT结果显示,沉默Stat3表达后的OV2008细胞与空白对照组以及Ad5/dE1A病毒对照组的细胞相比,增殖能力明显减弱（P<0.05）;Transwell小室法结果显示,沉默Stat3后的OV2008细胞12.64±7.10与空白对照组127.70±8.28以及Ad5/dE1A病毒对照组137.80±9.78相比,侵袭运动能力明显减弱（P<0.05）;流式细胞术检测结果显示,沉默Stat3表达后的OV2008细胞65.51±5.78与空白对照组10.73±4.28以及Ad5/dE1A病毒对照组12.17±3.87相比,细胞凋亡率明显增加（P<0.05）。结论：沉默Stat3能明显抑制卵巢癌细胞系OV2008的增殖,降低其侵袭运动能力,并诱导其凋亡,即沉默Stat3能降低卵巢癌细胞系OV2008的恶性表型,提示Stat3可作为卵巢癌基因治疗的一个新的分子靶点。     

Identification of two novel inhibitors of mTOR signaling pathway based on high content screening

Purpose

Mammalian target of rapamycin (mTOR) signaling pathway plays a critical role in regulating cell growth, proliferation and survival. Dysregulation of mTOR signaling pathway is closely involved in cancer development and chemotherapy resistance. Inhibitors of mTOR signaling pathway have been demonstrated to be attractive therapeutics for cancer therapy. In the present study, we aim to discover novel mTOR signaling pathway inhibitors from a natural compound library.

Methods

Inhibitors of mTOR signaling pathway were discovered via high content screen assay based on the subcellular localization of eukaryotic initiation factor 4E (eIF4E) in mouse embryonic fibroblast cells. Candidate compounds were further assessed in cancer cells. Phosphorylation levels of mTOR complexes downstream targets were analyzed using Western blot. Cell cytotoxicity and apoptosis were evaluated using MTS assay and flow cytometry, respectively.

Results

Two compounds, 1,4-O-diferuloylsecoisolariciresinol (IM-1) and Pierreione B (IM-2), were identified which induced significant nuclear translocation of eIF4E in a panel of cancer cells. Both of the compounds decreased the phosphorylation levels of p70 ribosomal protein S6 kinase (S6K) and eIF4E binding protein 1 (4E-BP1), resulting in cancer cell cytotoxicity and apoptosis.

Conclusions

Via high content screen assay, two novel inhibitors of mTOR signaling, IM-1 and IM-2, were identified with strong anticancer activity. IM-1 and IM-2 could be potential candidates for anticancer therapeutics by targeting mTOR signaling pathway and as such warrants further exploration.     

Atiprimod blocks phosphorylation of JAK-STAT and inhibits proliferation of acute myeloid leukemia (AML) cells

In studies of multiple myeloma cells, atiprimod was shown to block Stat3 activation and inhibited colony-forming cell proliferation. We hypothesized that atiprimod may also inhibit activation of intracellular signaling pathways in AML cells resulting in apoptosis and growth inhibition. We demonstrate that atiprimod inhibited clonogenic growth of AML cell lines and fresh AML marrow cells whereas it did not significantly affect growth of normal hematopoietic progenitors from marrow samples of healthy controls. Atiprimod decreased phosphorylation of Stat3 and Stat5, and protein levels of Jak2, whereas gene expression of Jak2 was not affected. Atiprimod further induced apoptosis by cleavage of caspase 3 and PARP. In summary, our data suggest that atiprimod has a significant antiproliferative and proapoptotic effect on AML cells. This effect may be facilitated by inhibition of the Jak-Stat signaling pathway. Further evaluation of atiprimod in clinical trials of AML should be considered.     

Selective inhibitors of nuclear export (SINE) as novel therapeutics for prostate cancer

Mislocalization of proteins is a common feature of cancer cells. Since localization of proteins is tightly linked to its function, cancer cells can inactivate function of a tumor suppressor protein through mislocalization. The nuclear exportin CRM1/XPO 1 is upregulated in many cancers. Targeting XPO 1 can lead to nuclear retention of cargo proteins such as p53, Foxo, and BRCA1 leading to cell cycle arrest and apoptosis. We demonstrate that selective inhibitors of nuclear export (SINE) can functionally inactivate XPO 1 in prostate cancer cells. Unlike the potent, but toxic, XPO 1 inhibitor leptomycin B, SINE inhibitors (KPT-185, KPT-330, and KPT-251) cause a decrease in XPO 1 protein level through the proteasomal pathway. Treatment of prostate cancer cells with SINE inhibitors lead to XPO 1 inhibition, as evaluated by RevGFP export assay, leading to nuclear retention of p53 and Foxo proteins, consequently, triggering apoptosis. Our data reveal that treatment with SINE inhibitors at nanomolar concentrations results in decrease in proliferation and colonogenic capacity of prostate cancer cells by triggering apoptosis without causing any cell cycle arrest. We further demonstrate that SINE inhibitors can be combined with other chemotherapeutics like doxorubicin to achieve enhanced growth inhibition of prostate cancer cells. Since SINE inhibitors offer increased bioavailability, reduced toxicity to normal cells, and are orally available they can serve as effective therapeutics against prostate cancer. In conclusion, our data reveals that nucleocytoplasmic transport in prostate cancer can be effectively targeted by SINE inhibitors.     

Synergistic cytotoxic effect of sulindac and pyrrolidine dithiocarbamate against ovarian cancer cells

Sulindac, a non-steroidal anti-inflammatory drug, suppresses carcinogenesis and inhibits growth of tumor cells. Pyrrolidine dithiocarbamate (PDTC), a potent NF-κB inhibitor, has been also identified as a potential anti-neoplastic agent. We hypothesized that combination of sulindac and PDTC could result in augmentation of cytotoxicity against ovarian cancer cells. The effect of sulindac and PDTC was examined on several ovarian cancer lines. Tumor cell viability was assessed using the MTT assay. Annexin-V/PI staining was used to detect apoptosis, cell cycle distribution was analyzed in FACS, and expression of cellular proteins was detected by western blotting. Incubation of OVA-14, OVP-10 and CAOV-1 ovarian cancer cells with sulindac and PDTC resulted in significantly greater inhibition of cell viability compared to either compound alone. In a model of OVA-14 cells it was evident that this effect was not related to the expression of COX enzymes since both active (sulindac sulfide) and inactive (sulindac) in vitro compounds affected the growth of tumor cells to a similar extent and synergized in cytotoxicity with PDTC. Combination of sulindac and PDTC lead to G0 arrest and massive apoptosis in co-treated cultures. Western blotting analysis argued for induction of the mitochondrial apoptotic pathway. These data demonstrate the synergistic cytotoxic effect of sulindac and PDTC on ovarian cancer cells through apoptosis and cell cycle arrest and prompt to test the efficacy of this combination in animal models.     

Favorable prognostic value of SOCS2 and IGF-I in breast cancer

Background  

Suppressor of cytokine signaling (SOCS) proteins comprise a protein family, which has initially been described as STAT induced inhibitors of the Jak/Stat pathway. Recent in vivo and in vitro studies suggest that SOCS proteins are also implicated in cancer. The STAT5 induced IGF-I acts as an endocrine and para/autocrine growth and differentiation factor in mammary gland development. Whereas high levels of circulating IGF-I have been associated with increased cancer risk, the role of autocrine acting IGF-I is less clear. The present study is aimed to elucidate the clinicopathological features associated with SOCS1, SOCS2, SOCS3, CIS and IGF-I expression in breast cancer.     

Hsp70 promotes chemoresistance by blocking Bax mitochondrial translocation in ovarian cancer cells

Cisplatin can induce apoptosis in ovarian cancer cells through the mitochondrial death pathway, and dysregulation of this pathway contributes to cisplatin resistance in ovarian cancer cells. Here we show that cisplatin induces mitochondrial proteins such as Smac/DIABLO, Cytochrome c, and HrtA2/Omi release to the cytosol and apoptosis in cisplatin-sensitive, but not -resistant ovarian cancer cells. Bax translocation to mitochondria is required for mitochondrial protein release and cisplatin-induced apoptosis in human ovarian cancer cells. Hsp70 is highly expressed in cisplatin-resistant cells. Hsp70 promotes chemoresistance, in part, by blocking Bax translocation to the mitochondria and mitochondrial protein release to cytosol in human ovarian cancer cells.     

角鲨烯环氧化酶对卵巢癌细胞生物学行为的影响及其机制探讨

目的:观察角鲨烯环氧化酶（squalene epoxidase,SQLE）对卵巢癌细胞增殖、迁移、侵袭和凋亡的影响,并从上皮间质转化(epithelial mesenchymal transformation,EMT)方向探究其可能的机制。方法:通过搜索GEPIA和Western blotting、qRT-PCR确定SQLE是否存在差异表达;Kaplan-Meier Plotter在线网站评估SQLE与卵巢癌预后的关系;构建稳定敲减SQLE的A2780细胞株和稳定过表达SQLE的ES-2细胞株,Western blotting、qRT-PCR检测敲减和过表达效率;MTT和集落克隆实验、划痕实验及Transwell小室分别用于检测细胞增殖、迁移及侵袭能力的变化;流式细胞术检测凋亡变化;Western blotting检测Ki67、EMT、凋亡、Wnt/β-catenin信号通路相关蛋白的表达。结果:SQLE在卵巢癌组织和细胞中表达水平明显高于正常卵巢组织和细胞(P＜0.05);SQLE表达水平与卵巢癌预后明显相关(P＜0.05);敲减SQLE后细胞增殖、迁移及侵袭能力减弱(P＜0.05)、细胞凋亡增加(P＜0.05),并导致Ki67、EMT、凋亡以及Wnt/β-catenin信号通路相关蛋白的变化(P＜0.05),过表达SQLE时趋势则相反;XAV-939可逆转过表达SQLE的ES-2细胞中相关蛋白表达水平的变化。结论:SQLE可能通过激活Wnt/β-catenin信号通路增强卵巢癌细胞的增殖、迁移及侵袭能力,抑制其凋亡,并促进上皮间质转化。     

Jak/Stat pathway in Waldenström's macroglobulinemia

While high serum concentrations of immunoglobulin M (IgM) protein are responsible for much of the morbidity associated with Waldenström's macroglobulinemia (WM), the mechanisms controlling IgM secretion in this malignancy remain unknown. The Jak/Stat signaling pathway mediates the effects of most cytokines involved in stimulating immunoglobulin production in nonmalignant B cells. However, in many hematopoietic malignancies, cytokine-driven stimulation of the Jak/Stat pathway is hyperactive, promoting the expression of genes associated with proliferation and survival. Autocrine/paracrine signaling of cytokines present in the tumor microenvironment may lead to persistent Jak/Stat signal transduction in WM as well. Yet despite evidence implicating the Jak/Stat pathway in the pathogenesis of WM, the relationship between Jak/Stat-mediated cytokine signaling and IgM hypersecretion has not been defined. Through the use of targeted Jak/Stat inhibitors, it is now possible to examine the contribution of aberrant cytokine signaling to IgM production, potentially allowing for a more targeted approach to WM therapy.