WP1066 Treatment Research Articles

CRISPR-mediated WNK4 point mutation aggravates tumor progression and weakens chemotherapy sensitivity in gastric cancer.

Gastric cancer (GC) is the fifth most common malignancy, the molecular targets of which have been increasingly explored in recent years. As a serine/threonine protein kinase, the role of WNK lysine deficient protein kinase 4 (WNK4) in GC was clarified in this study. Human GC lines AGS and MKN45 were stably transfected with a WNK4 mutant constructed by the CRISPR/Cas9 method and treated with cis-dichlorodiammine platinum (CDDP, 2 μg/mL) and 5-fluorouracil (5-FU, 5 μg/mL) for 48h. Tumor-bearing mice were established with 5×106 mutant-type AGS cells, and injected with 40 mg/kg WP1066, the inhibitor of signal transducer and activator of transcription 3 (STAT3), for 21 days. Cell malignant potential and tumor growth were assessed. STAT3 activation was identified by western blot and immunohistochemistry. The interaction between WNK4 and STAT3 was determined using co-immunoprecipitation and immunofluorescence co-localization. WNK4 mutation promoted proliferation and invasion, and upregulated the p-STAT3/STAT3 value in GC cells with/without 5-FU and CDDP treatments, while inhibiting apoptosis of GC cells without drug treatment. In tumor-bearing mice, WNK4 mutation accelerated tumor growth, increased levels of p-STAT3, STAT3, and p-STAT3/STAT3, and strengthened the co-immunoprecipitation and co-localizing with STAT3; however, these effects were reversed by WP1066 treatment. Through activating STAT3, WNK4 mutation impacts both the natural and drug-treated growth of GC cells or tumors, suggesting a new avenue for preclinical research.

MiR-141-3p Regulates the Proliferation and Apoptosis of Endometrial-Myometrial Interface Smooth Muscle Cells in Adenomyosis Via JAK2/STAT3 Pathway.

Adenomyosis (ADS) is a common benign gynecological disease. Abnormal proliferation at the endometrial-myometrial interface (EMI) plays a crucial role in the occurrence and progression of ADS. miR-141-3p is associated with cell proliferation and apoptosis. However, the specific mechanism of miR-141-3p in the etiology of ADS is still unknown. In this study, we explored the effects of miR-141-3p on the proliferation and apoptosis of ADS EMI smooth muscle cells (SMCs). We collected EMI tissues for the primary culture of SMCs from 25 patients diagnosed with ADS and 20 without ADS. Real-time quantitative polymerase chain reaction and western blot were used to measure the mRNA and protein expression levels of miR-141-3p, JAK2, STAT3, phospho-JAK2, and phospho-STAT3 in ADS EMI SMCs. The cell counting kit 8 assay and flow cytometry analysis were used to evaluate the proliferation and apoptosis of EMI SMCs. The miR-141-3p mimic/inhibitor was used to increase or decrease the expression level of miR-141-3p. We added WP1066 to block the phosphorylation of JAK2/STAT3 pathway components. The miR-141-3p levels were decreased, while JAK2 and STAT3 levels were increased in ADS EMI SMCs. miR-141-3p overexpression significantly inhibited the proliferation and enhanced the apoptosis of EMI SMCs, whereas a decrease in miR-141-3p expression level was connected to the opposite results. Meanwhile, inactivated JAK2/STAT3 pathway decreased proliferation and enhanced apoptosis of EMI SMCs after WP1066 treatment. Furthermore, rescue experiments confirmed that the JAK2/STAT3 pathway was the downstream pathway of miR-141-3p and reduced the effect of miR-141-3p on the proliferation and apoptosis of EMI SMCs. These results demonstrate that miR-141-3p regulates the proliferation and apoptosis of ADS EMI SMCs by modulating the JAK2/STAT3 pathway.

Dexmedetomidine inhibits apoptosis of astrocytes induced by oxygen-glucose deprivation via targeting JAK/STAT3 signal pathway

ObjectiveThere is an increasing interest concerning the contribution of astrocytes to the intrinsic bioremediation of ischemic brain injury. The aim of this work was to disclose the effects and mechanism of dexmedetomidine (DEX) on the apoptosis of astrocytes under oxygen glucose deprivation (OGD) condition. MethodsPrimary cultured astrocytes separated from Sprague-Dawley (SD) rats were subjected to OGD treatment. Astrocytes were transfected with si-JMJD3 or pcDNA3.1-JMJD3 and then treated with DEX or JAK/STAT inhibitor (WP1066) before cell apoptosis was detected by TUNEL apoptosis kit. Western blot was applied to assess the level of apoptosis-related proteins Caspase-3, Bax and Bcl-2. Astrocyte cell viability was assessed by measuring the lactate dehydrogenase (LDH) level using a LDH assay kit. ResultsAstrocytes received OGD treatment had increased LDH and elevated apoptotic rate (P < 0.05). DEX could suppress OGD induced cytotoxic effect on astrocytes, as evidenced by decreased LDH release and suppressed cell apoptosis rate (P < 0.05). Meanwhile, DEX and WP1066 treatment were also found to inhibit the phosphorylation level of STAT1 and STAT3 (P < 0.05), indicating the DEX could suppress the activation of JAK/STAT signal pathway. JMJD3 overexpression in astrocytes could suppress the anti-apoptotic function of WP1066 in OGD treated astrocytes and hamper the protective effect of DEX in cell apoptosis (P < 0.05), suggesting that DEX and JAK/STAT signal pathway inhibits OGD induced apoptosis in astrocytes by down-regulating JMJD3. ConclusionDEX protects astrocytes against apoptosis via inhibiting JAK2/STAT3 signal pathway and downregulating JMJD3 expression in vitro.

Stat3 inhibitor abrogates the expression of PD-1 ligands on lymphoma cell lines.

Recent studies have indicated the significance of immune checkpoint molecules including programmed death-1 (PD-1), cytotoxic T-lymphocyte associated protein 4, and T-cell immunoglobulin and mucin domain-containing molecule-3 for anti-tumor immune responses. We previously investigated PD-1 ligand 1/2 (PD-L1/2) expression in lymphoma cell lines, and found that PD-L1/2 is expressed on the adult T-cell leukemia/lymphoma (ATL-T) and B-cell lymphoma (SLVL) cell lines. In the present study, we investigated whether the Stat3 inhibitor WP1066 abrogated PD-L1/2 expression in lymphoma cell lines. Incubation with WP1066 inhibited lymphoma cell growth and induced cell apoptosis. PD-L1/2 expression in the ATL-T, SLVL, and human brain malignant lymphoma (HKBML) cell lines was significantly abrogated by WP1066 treatment. These data indicated that a Stat3 inhibitor abrogated PD-L1/2 expression in lymphoma cells. Such an inhibitor is therefore considered to be useful for additional immunotherapy in patients with advanced lymphoma.

STAT3 signaling pathway regulates glioma stem cells induced host macrophage malignance

Background: Continuous activation of the signal transducer and activator of transcription 3 (STAT3) signaling pathway contributes to cellular proliferation, angiogenesis and activation of anti-apoptosis pathways to promote tumor progression. Besides, STAT3 activation plays an important role in the polarisation of M2 macrophages and immune suppression. Our previous study found that glioma stem cells (SU3) could induce host macrophage malignance in the immune microenvironment, and a cancerous cell line SU3-induced host celiac tumor cells (SU3-ihCTCs) was obtained. We also found STAT3 was activated in SU3-ihCTCs. Methods: The ratio of F4/80 positive cells in SU3-ihCTCs was tested by flow cytometry, and the expression of p-STAT3 and macrophage markers were analyzed with RT-PCR or Western Blotting, respectively. SU3-ihCTCs were treated with 1–6 μmol/L WP1066 for 24 hours, then the expression of STAT3, p-STAT3, Bcl-2 and Cleaved caspase-3 were detected with Western Blotting, and the ratio of apoptosis were tested by flow cytometry. BalB/C nude mice received subcutaneous implants of SU3-ihCTCs, the tumor size was measured every other day, and the weight of tumor was measured. The expression of STAT3, p-STAT3, Bcl-2 and Cleaved caspase3 of the transplanted tumor were tested by Western Blotting and immunohistochemical analysis. Results: SU3-ihCTCs expressed higher of p-STAT3 and M2 macrophage makers. When SU3-ihCTCs were treated with the STAT3 inhibitor WP1066, the expressions of M2 makers, p-STAT3 and Bcl-2 of the cells were decreased, and the expression of cleaved caspase-3 was increased. At the same time, the proliferation of SU3-ihCTCs was inhibited and apoptosis was enhanced. In vivo studies had shown that WP1066 treatment significantly decreased the volume and weight of SU3-ihCTCs xenografts in nude mice, and immunohistochemical and Western blotting analyses showed that the expression levels of p-STAT3 and Bcl-2 decreased and those of the apoptosis-related protein caspase-3 were upregulated. Conclusions: We suggest that glioma stem cells induce host macrophage malignance in tumor microenvironment (TME), STAT3 pathway play an important role in the transformation process. These results present new evidence that macrophages in TME promote tumor proliferation. STAT3 signaling pathway may present a therapeutic target to suppress the tumor development.

Abstract 3196: STAT3 inhibitor WP1066 as a novel therapeutic for medulloblastoma

Abstract The purpose of this study is to determine the efficacy of the STAT3 inhibitor WP1066 on the sonic-hedgehog (SHH) subtype of medulloblastoma (MB). STAT3, or signal transducer and activator of transcription 3, is a potential novel target for the treatment of SHH-MB. We have generated substantial preliminary data showing that STAT3 activation promotes the survival of SHH-MB cancer stem cells (CSCs) that are critical for treatment resistance and tumor recurrence. Smoothened (SMO) antagonists have shown efficacy in treating subtypes of SHH-MB, however all tumors develop resistance and SMO inhibitors are ineffective for patients with SHH-MB harboring mutations downstream of SMO. Our preliminary in vitro data suggests that combined SMO and STAT3 inhibition has synergistic activity, and that constitutively activated STAT3 is protective against SHH-MB cell death mediated by SMO inhibition. Methods: The effect of increasing concentrations of WP1066 on Shh-responsive MB cells was assessed by Western blot analysis for STAT3 phosphorylation, WST assay for cell proliferation, and soft agar colony formation. The effect of WP1066 on apoptosis and the cell cycle was assessed by flow cytometry. An ex vivo murine SmoA1 SHH-MB brain slice culture method was used to assess the effects of WP1066 alone and in combination with radiation. In vivo studies consist of mice treated with 1) WP1066, 2) SMO inhibitor LDE225 3) WP1066 in combination with LDE225 and 4) vehicle alone. All mice are imaged by MRI prior to treatment and every 3 weeks after treatment. Tumor volume is calculated and rate of tumor growth and survival are measured. The mice are followed until death or the end of the experiment at 36 weeks. Results: In human Shh-responsive MB cells in vitro, WP1066 blocked IL6-induced STAT3 activation (phosphorylation), decreased cell proliferation and abrogated tumor colony formation. Results regarding the mechanism of inhibition of cell proliferation/viability by WP1066 (i.e. apoptosis and cell cycle analysis) are pending. In the SHH-MB brain tumor slice cultures, WP1066 treatment induced potent tumor cell kill, especially of the CSCs within the perivascular niche. WP1066 also showed synergy with radiation, but did not add toxicity to normal brain cells. Preliminary in vivo data indicate a statistically significant decrease in the rate of tumor growth and prolonged survival in mice treated with WP1066 compared to vehicle control. Results of the efficacy of WP1066 with LDE225 are pending. Conclusions: WP1066 has potent efficacy against Shh-responsive MB cells in vitro and SHH-MB tumors ex vivo, without toxicity to normal brain cells. Preliminary data is promising for WP1066 activity against SHH-MB in vivo, suggesting WP1066 is a novel therapeutic for SHH-MB. Citation Format: Laura K. Metrock, Jingbo Liu, Liangping Yuan, Hongying Zhang, Abhinav Dey, Tobey MacDonald. STAT3 inhibitor WP1066 as a novel therapeutic for medulloblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3196.

Delayed Administration of WP1066, an STAT3 Inhibitor, Ameliorates Radiation-Induced Lung Injury in Mice.

The present study was designed to investigate the effects of WP1066, a specific inhibitor of STAT3 signaling, on radiation-induced lung injury in mice. C57BL/6J mice were subjected to a single thoracic irradiation of 15 Gy X-ray and WP1066 was administrated through intraperitoneal injection. The early and delayed treatment groups were treated with WP1066 during the first 2 weeks and the second 2 weeks, respectively. The therapeutic effects of WP1066 were evaluated by survival analysis, histological examination, and measurement of inflammatory parameters and collagen deposition. The activation of STAT3 pathway was also estimated by immunohistochemical staining and Western blotting. Delayed treatment of WP1066, but not early treatment, prolonged survival time and prevented the development of radiation pneumonitis and the subsequent lung fibrosis in mice. WP1066 treatment also significantly suppressed the activation of STAT3 signaling in the irradiated lung tissues. The activation of STAT3 pathway might play an important part in the pathogenesis of radiation-induced lung injury. The protective effects of delayed treatment of WP1066 suggested STAT3 signaling could be a therapeutic target for radiation pneumonitis.

Overcoming Chemoresistance of Pediatric Ependymoma by Inhibition of STAT3 Signaling

The long-term clinical outcome of pediatric intracranial epepdymoma is poor with a high rate of recurrence. One of the main reasons for this poor outcome is the tumor’s inherent resistance to chemotherapy. Signal transducer and activator of transcription 3 (STAT3) is overactive in many human cancers, and inhibition of STAT3 signaling is an emerging area of interest in oncology. In this study, the possibility of STAT3 inhibition as a treatment was investigated in pediatric intracranial ependymoma tissues and cell lines. STAT3 activation status was checked in ependymoma tissues. The responses to conventional chemotherapeutic agents and a STAT3 inhibitor WP1066 in primarily cultured ependymoma cells were measured by cell viability assay. Apoptosis assays were conducted to reveal the cytotoxic mechanism of applied agents. Knockdown of STAT3 was tried to confirm the effects of STAT3 inhibition in ependymoma cells. High levels of phospho-STAT3 (p-STAT3) expression were observed in ependymoma tissue, especially in the anaplastic histology group. There was no cytotoxic effect of cisplatin, ifosfamide, and etoposide. Both brain tumor-initiating cells (BTICs) and bulk tumor cells (BCs) showed considerably decreased viability after WP1066 treatment. However, BTICs had fewer responses than BCs. No additive or synergistic effect was observed for combination therapy of WP1066 and cisplatin. WP1066 effectively abrogated p-STAT3 expression. An increased apoptosis and decreased Survivin expression were observed after WP1066 treatment. Knockdown of STAT3 also decreased cell survival, supporting the critical role of STAT3 in sustaining ependymoma cells. In this study, we observed a cytotoxic effect of STAT3 inhibitor on ependymoma BTICs and BCs. There is urgent need to develop new therapeutic agents for pediatric ependymoma. STAT3 inhibitors may be a new group of drugs for clinical application.

JAK/STAT pathway regulation of GABAA receptor expression after differing severities of experimental TBI

Synaptic inhibition in the adult brain is primarily mediated by the γ-aminobutyric acid (GABA) type A receptor (GABAAR). The distribution, properties, and dynamics of these receptors are largely determined by their subunit composition. Alteration of subunit composition after a traumatic brain injury (TBI) may result in abnormal increased synaptic firing and possibly contribute to injury-related pathology. Several studies have shown that the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathway can alter GABAAR subunit expression. The present study investigated changes in JAK/STAT pathway activation after two different severities of experimental TBI in the mouse using the controlled cortical impact (CCI) model. It also investigated whether modulating the activation of the JAK/STAT pathway after severe controlled cortical impact (CCI-S) with a JAK/STAT inhibitor (WP1066) alters post-traumatic epilepsy development and/or neurological recovery after injury. Our results demonstrated differential changes in both the activation of STAT3 and the expression of the GABAAR α1 and γ2 subunit levels that were dependent on the severity of the injury. The change in the GABAAR α1 subunit levels appeared to be at least partly transcriptionally mediated. We were able to selectively reverse the decrease in GABAAR α1 protein levels with WP1066 treatment after CCI injury. WP1066 treatment also improved the degree of recovery of vestibular motor function after injury. These findings suggest that the magnitude of JAK/STAT pathway activation and GABAAR α1 subunit level decrease is dependent on injury severity in this mouse model of TBI. In addition, reducing JAK/STAT pathway activation after severe experimental TBI reverses the decrease in the GABAAR α1 protein levels and improves vestibular motor recovery.

Inhibitor of signal transducer and activator of transcription 3 (STAT3) suppresses ovarian cancer growth, migration and invasion and enhances the effect of cisplatin in vitro.

The aim of the present study was to investigate the anti-ovarian cancer effect of the inhibitor of signal transducer and activator of transcription 3 (STAT3), WP1066. Western blot was used to detect the phosphorylation of STAT3 in ovarian cancer cell line SKOV3 and cisplatin-resistant ovarian cancer cell line SKOV3/DDP. MTT and colony-forming assays were performed to evaluate the viability and growth of ovarian cancer cells. The apoptosis of ovarian cancer cells was determined by flow cytometry. The wound healing assay and Transwell assay were performed to examine the migration and invasion of ovarian cancer cells. WP1066 significantly inhibited the phosphorylation of STAT3 in SKOV3 and SKOV3/DDP cells. WP1066 treatment inhibited the proliferation and clonogenicity of both SKOV3 and SKOV3/DDP cells. After WP1066 treatment for 24 h, the apoptosis rates of SKOV3 and SKOV3/DDP cells were significantly increased compared with the control cells. After treatment with WP1066, the reduction of the wound gaps was significantly less in both SKOV3 and SKOV3/DDP cells. WP1066 also significantly inhibited the invasion capacity of SKOV3 and SKOV3/DDP cells compared with the control group. Treatment with WP1066 combined with cisplatin significantly increased proliferation inhibition and apoptosis in SKOV3 and SKOV3/ DDP cells compared with treatment with cisplatin alone. A synergistic action between WP1066 and cisplatin on the proliferation and apoptosis of ovarian cancer cells was determined. In conclusion, inhibition of STAT3 may suppress the proliferation, migration and invasion, induce apoptosis and enhance the chemosensitivity of ovarian cancer cells, indicating that STAT3 is a new therapeutic target of ovarian cancer.

Abstract 17436: STAT3 Inhibition Protects Against Cardiomyocyte Hypertrophy Through a Novel NF-kB p65/Nrf2/HO-1 Pathway

Introduction: Left ventricular hypertrophy is the end-result of diverse cardiovascular pathologies. STAT3 signaling has been implicated in cardiomyocyte hypertrophy, although the precise mechanism remains incompletely understood. Hypothesis: We hypothesized that STAT3 induces myocyte hypertrophy via NF-kB/Nrf2 pathway. Methods and Results: Treatment of H9c2 cells with 1 μM Angiotensin II for 48 h induced a 2.4-fold increase in cell size compared with controls. This cellular hypertrophy was significantly inhibited in the presence of STAT3 inhibitor WP1066 (Figure). The levels of heme oxygenase-1 (HO-1), a key molecule in cardioprotective signaling, were increased by WP1066 treatment and decreased by STAT3 overexpression. Further investigation of the underlying mechanism revealed that WP1066 blocks the inhibitory effect of NF-kB p65 on Nrf2, leading to an upregulation of HO-1 (Figure). By using chromosome-IP and bioinformatics analysis, we identified that p65 suppresses Nrf2 expression by directly binding to the promoter region of Nrf2. The p65/Nrf2/HO-1 signaling pathway was further confirmed by using both p65 transient overexpression and NF-kB inhibitor pyrrolidine dithiocarbamate (PDTC). Conclusions: We conclude that angiotensin II-induced cardiomyocyte hypertrophy involves STAT3 signaling and downstream components include p65, Nrf2, and HO-1. These novel molecular insights may lead to development of new therapy to prevent and treat cardiac hypertrophy.

STAT3 inhibitor WP1066 attenuates miRNA-21 to suppress human oral squamous cell carcinoma growth in vitro and in vivo

Abnormalities in signal transducer and activator of transcription 3 (STAT3) are involved in the oncogenesis of oral squamous cell carcinoma (OSCC). MicroRNA-21 (miR-21) is an important gene expression regulator to OSCC. miR-21 induction by STAT3 has been reported in multiple human cancers. In the present study, we found that STAT3 (-/p) expression was positively correlated with miR-21 in 60 OSCC samples. A reporter gene assay showed that miR-21 overexpression was dependent on STAT3 activation. WP1066, a small molecular inhibitor of STAT3, was used to suppress STAT3 expression in OSCC cells. TSCCA and TCA8113 showed reduction in tumor cell proliferation, invasion ability and miR-21 expression by WP1066 treatment. In addition, the expression of miR-21 target proteins [programmed cell death 4 (PDCD4), tissue inhibitor of metalloproteinase 3 (TIMP-3) and phosphatase and tensin homolog (PTEN)] was upregulated. Restored STAT3 expression by IL-6 induced miR-21 overexpression, which further confirmed the correlation between STAT3 and miR-21. WP1066 inhibited tumor growth and induced tumor cell apoptosis in the TSCCA xenograft tumor model. Western blotting and immunohistochemistry staining indicated that STAT3 (-/p), Ki67, Bcl-2 and MMP-2 expressions decreased in the WP1066-treated group; PDCD4, TIMP-3 and PTEN expression increased simulta-neously. The present study provides evidence that targeting STAT3 could regulate OSCC cell growth in a miR-21-dependent manner and WP1066 could be a novel candidate drug to treat OSCC by inhibiting STAT3/miR-21 axis.

Abstract B211: Augmentation of Fas (CD95) signaling pathway sensitizes non-small cell lung cancer (NSCLC) cells.

Abstract Epidermal growth factor receptor (EGFR) is a proven therapeutic target to treat a subset of NSCLC harboring activating mutations within the EGFR gene. However, many NSCLC patients are not sensitive to EGFR kinase inhibitors, suggesting that other factors may play a role in determining survival of NSCLC cells. Signal transducers and activators of transcription 3 (Stat3) functions as a transcription factor to mediate cell survival and differentiation and the dysregulation of Stat3 has been described in numbers of cancers. Our previous report showed that suppression of Stat3 activity by a pharmacological approach, WP1066, sensitized NSCLC cells. Along with Stat3 inhibition, we also found that WP1066 treatment up-regulated the expression of Fas, a death receptor, regulating numbers of physiological and pathological process of cell death. Although Fas-mediated apoptosis pathway has been studied mostly in the immune system, the identification of Fas mutations in non-lymphoid malignancies, such as NSCLC, suggests its implication in the pathogenesis of non-lymphoid malignancies as well. Results from this study showed that levels of Fas were increased in cells with down-regulated Stat3 by small interfering RNA (siRNA), suggesting that Stat3 may play an inhibitory role to regulate the expression of Fas. To examine whether the Fas pathway is responsible for the WP1066-mediated cytotoxicity, Fas apoptosis signaling was induced by anti-Fas antibody and Fas ligand. Our results revealed that NSCLC cells showed different degrees of sensitivity to anti-Fas antibody but resistance to Fas ligand. However, augmentation of Fas expression by WP1066 remarkably increased the sensitivity of NSCLC cells to both anti-Fas antibody and Fas ligand. Taken together, our findings suggest that regulation of the Fas system may be a potential strategy to treat NSCLC and the combined use of a Fas inducer, such as WP1066, may further strengthen its efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B211.

A novel inhibitor of the STAT3 pathway induces apoptosis in malignant glioma cells both in vitro and in vivo

Signal transducer and activator of transcription-3 (STAT3) is constitutively activated in a variety of cancer types, including malignant gliomas. STAT3 is activated by phosphorylation of a tyrosine residue, after which it dimerizes and translocates into the nucleus. There it regulates the expression of several genes responsible for proliferation and survival at the transcriptional level. A selective inhibitor of STAT3 phosphorylation, AG490, has been shown to inhibit growth and induce apoptosis in some cancer cell types. However, although AG490 routinely shows in vitro anticancer activity, it has not consistently demonstrated an in vivo anticancer effect in animal models. Here, we have tested WP1066, a novel inhibitor structurally related to AG490 but significantly more potent and active, against human malignant glioma U87-MG and U373-MG cells in vitro and in vivo. IC(50) values for WP1066 were 5.6 muM in U87-MG cells and 3.7 muM in U373-MG cells, which represents 18-fold and eightfold increases in potency, respectively, over that of AG490. WP1066 activated Bax, suppressed the expression of c-myc, Bcl-X(L) and Mcl-1, and induced apoptosis. Systemic intraperitoneal administration of WP1066 in mice significantly (P<0.001) inhibited the growth of subcutaneous malignant glioma xenografts during the 30-day follow-up period. Immunohistochemical analysis of the excised tumors revealed that phosphorylated STAT3 levels in the WP1066 treatment group remained inhibited at 3 weeks after the final WP1066 injection, whereas tumors from the control group expressed high levels of phosphorylated STAT3. We conclude that WP1066 holds promise as a therapeutic agent against malignant gliomas.