[Therapeutic mechanism of basic fibroblast growth factor on spinal cord injury in rats based on the Notch/signal transducer and activator of transcription 3 signaling pathway].

Objective: To explore the therapeutic effect of basic fibroblast growth factor (bFGF) on spinal cord injury (SCI) in rats and the influence of Notch/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Methods: A total of 40 10-week-old male Sprague Dawley (SD) rats were selected to establish T 10-segment SCI model by a free falling object. Among them, 32 successful models were randomly divided into model group and bFGF group, with 16 in each group. Another 16 SD rats were selected as sham-operation group, with only T 10 processes, dura mater, and spinal cord exposed. After modeling, the rats in bFGF group were intraperitoneally injected with 100 µg/kg bFGF (once a day for 28 days), and the rats in model group and sham-operation group were injected with normal saline in the same way. The survival of rats in each group were observed after modeling. Basso-Beattie-Bresnahan (BBB) scores were performed before modeling and at immediate, 14 days, and 28 days after modeling to evaluate the functional recovery of hind limbs. Then, the spinal cord tissue at the site of injury was taken at 28 days and stained with HE, Nissl, and propidium iodide (PI) to observe the pathological changes, neuronal survival (number of Nissl bodies) and apoptosis (number of PI red stained cells) of the spinal cord tissue; immunohistochemical staining and ELISA were used to detect the levels of astrocyte activation markers [glial fibrillary acidic protein (GFAP)] and inflammatory factors [interleukin 1ß (IL-1ß), tumor necrosis factor α (TNF-α), interferon γ (IFN-γ)] in tissues, respectively. Western blot was used to detect the expressions of Notch/STAT3 signaling pathway related proteins [Notch, STAT3, phosphoryl-STAT3 (p-STAT3), bone morphogenetic protein 2 (BMP-2)] in tissues. Results: All rats survived until the experiment was completed. At immediate after modeling, the BBB scores in model group and bFGF group significantly decreased when compared to sham-operation group ( P<0.05). At 14 and 28 days after modeling, the BBB scores in model group significantly decreased when compared to sham-operation group ( P<0.05); the bFGF group showed an increase compared to model group ( P<0.05). Compared with before modeling, the BBB scores of model group and bFGF group decreased at immediate after modeling, and gradually increased at 14 and 28 days, the differences between different time points were significant ( P<0.05). The structure of spinal cord tissue in sham-operation group was normal; in model group, there were more necrotic lesions in the spinal cord tissue and fewer Nissl bodies with normal structures; the number of necrotic lesions in the spinal cord tissue of the bFGF group significantly reduced compared to the model group, and some normally structured Nissl bodies were visible. Compared with sham-operation group, the number of Nissl bodies in spinal cord tissue significantly decreased, the number of PI red stained cells, GFAP, IL-1ß, TNF-α, IFN-γ, Notch, p-STAT3 /STAT3, BMP-2 protein expression levels significantly increased in model group ( P<0.05). The above indexes in bFGF group significantly improved when compared with model group ( P<0.05). Conclusion: bFGF can improve motor function and pathological injury repair of spinal cord tissue in SCI rats, improve neuronal survival, and inhibit neuronal apoptosis, excessive activation of astrocytes in spinal cord tissue and inflammatory response, the mechanism of which may be related to the decreased activity of Notch/STAT3 signaling pathway.

Antitumor effect of toosendanin on oral squamous cell carcinoma via suppression of p-STAT3.

BACKGROUND: Toosendanin (TSN) exhibits potent antitumor activity against various tumor cell lines. However, its efficacy against oral squamous cell carcinoma (OSCC) remains unknown. Here, we investigated the effects of TSN on OSCC cells in vitro and verified them in vivo using a patient-derived xenograft (PDX) model. METHODS: The effect of TSN on OSCC cells was investigated by cytotoxicity assays and flow cytometry. The expression of proteins was detected by western blotting. An OSCC PDX model was constructed to further investigate the role of TSN in regulating the function of OSCC. RESULTS: The cell viability of CAL27 and HN6 cells decreased as the concentration of TSN increased within the experimental range. Compared with controls, TSN at lower doses inhibited cell proliferation and induced apoptosis through S-phase cell cycle arrest. TSN inhibited OSCC cell proliferation by downregulating the STAT3 pathway through the inhibition of STAT3 phosphorylation. After successful construction of the OSCC PDX model with high pathological homology to the primary tumor and treatment with an intraperitoneal injection of TSN, we showed that TSN significantly reduced the tumor size of the PDX model mice without obvious toxicity. CONCLUSIONS: Both in vitro and in vivo, TSN significantly inhibits the proliferation and promoted apoptosis of OSCC cells. Furthermore, TSN demonstrates potent inhibition of STAT3 phosphorylation, indicating its potential as a promising therapeutic agent for OSCC. Therefore, TSN holds great promise as a viable drug candidate for the treatment of OSCC.

The prognostic value of expressions of STAT3, PD-L1, and PD-L2 in Ta/T1 urothelial carcinoma before and after BCG treatment.

INTRODUCTION: Nonmuscle invasive bladder cancers (NMIBC) are common tumors diagnosed in older individuals and men (median age: 69 years). Immunotherapy is a treatment option in cases resistant to Bacillus-Calmette-Guerin (BCG) therapy. We aimed to evaluate the prognostic role of programmed-cell-death ligand (PD-L)-1 (PD-L1), PD-L2, and signal transducer and activator of transcription 3 (STAT3) expressions, which are closely related to immune mechanisms, in the response to BCG treatment of NMIBC. METHODS: The data of patients at the Ta and T1 stages of the cancer without muscularis propria invasion, who were treated with intravesical BCG therapy between 2017 and 2022 were retrospectively analyzed. Immunohistochemical staining for PD-L1, PD-L2, and STAT3 was performed on transurethral resection materials. RESULTS: The mean age of 59 patients was 66.5 ±7.7 and 83.9% were male. The percentage of patients with complete response to BCG treatment was 66.1% and that of BCG refractory patients was 33.9%. Demographic and clinical data did not differ significantly according to BCG treatment response (P> 0.05). The proportion of patients with tumor-infiltrating lymphocytes (TILs) ≥20% were 9.7% among those with Ta-stage tumors and 46.4% among those with T1-stage tumors (P = 0.0014). The percentages of tumor cell scoring (TCS), immune cell scoring (ICS), combined scoring (CS), and expression levels of PD-L1, PD-L2, and H-score of STAT3 did not differ significantly according to tumor stage and treatment response (P > 0.05). However, the median ICS and CS for PD-L1 and median H-score for STAT3 were significantly higher among patients in T1 stage compared to those in Ta stage (P = 0.0487, 0.0462, 0.0112, respectively). Among BCG refractory patients, median STAT3 of patients in T1 stage was significantly higher than those at stage Ta (P = 0.0356) and the rate of TILs was ≥20% in only 3 patients in T1 stage (P = 0.031). Among all patients, significant positive correlation was found between TCS for PD-L1 and H-score for STAT3 (P = 0.0302); and between ICS for PD-L1 and TCS for PD-L2 (P = 0.0053) and TILs ratio (P < 0.0001). Among BCG-refractory cases, pretreatment and post-treatment TCS for PD-L2 and H-scores for STAT3 were significantly correlated (P = 0.0361 and 0.0021, respectively). CONCLUSIONS: The success of BCG treatment in NMIBC was not related to PD-L1, PD-L2, and STAT3 expression status, but PD-L1 expression was correlated with both PD-L2 and STAT3 as well as TILs rate, but this correlation was lost after BCG treatment.

A Dual-Targeting Liposome Enhances Triple-Negative Breast Cancer Chemoimmunotherapy through Inducing Immunogenic Cell Death and Inhibiting STAT3 Activation.

Immunotherapy gains increasing focus in treating triple-negative breast cancer (TNBC), while its efficacy is greatly restricted owing to low tumor immunogenicity and immunosuppressive tumor microenvironment (ITM). Herein, a LyP-1 and chondroitin sulfate (CS) dual-modified liposome co-loaded with paclitaxel (PTX) and cryptotanshinone (CTS), namely CS/LyP-1-PC Lip, is engineered for TNBC chemoimmunotherapy via induction of immunogenic cell death (ICD) and inhibition of signal transducer and activator of transcript-3 (STAT3) activation. CS/LyP-1-PC Lip enhances cellular uptake through p32 and CD44 dual receptor-mediated endocytosis. Within the tumor, the CS layer is continuously detached by hyaluronidase to release drugs. Subsequently, CTS sensitizes the cytotoxicity of PTX to 4T1 tumor cells. PTX induces ICD of tumor cells and facilitates infiltration of cytotoxic T lymphocyte to provoke immune response. Meanwhile, the concomitant delivery of CTS inhibits STAT3 activation to decrease infiltration of regulatory T cell, M2-type tumor-associated macrophage, and myeloid-derived suppressor cell, thus reversing ITM. Markedly, the dual-targeting liposome shows superior anti-tumor efficacy in subcutaneous TNBC mice and significant lung metastasis suppression in tumor metastasis model. Overall, this work offers a feasible combination regimen and a promising nanoplatform for the development of TNBC chemoimmunotherapy.

Targeting DCLK1 attenuates tumor stemness and evokes antitumor immunity in triple-negative breast cancer by inhibiting IL-6/STAT3 signaling.

Triple-negative breast cancer (TNBC) exhibits the poorest outcomes among breast cancer subtypes due to the high heterogeneity and a lasting scarcity of effectual treatments. Targeted therapies based on molecular subtypes of TNBC are critical step toward tailoring treatments to improve clinical outcomes. Gastrointestinal cancer stem cell (CSC) marker DCLK1 was reported to be highly expressed in stem cell-rich subtype of TNBC. Here, we firstly explored the impacts of DCLK1 on tumor cells as well as their immune microenvironment in TNBC and potential therapeutic strategies for TNBC patients with high DCLK1 expression. Our results disclosed that DCLK1 overexpression promoted, while knockout of DCLK1 suppressed the CSC-like traits of TNBC cells and resistance to chemotherapeutics. Besides, DCLK1 supported immune escape by inhibiting intratumoral cytotoxic T cell infiltration in TNBC and hence limited immune checkpoint inhibitors efficacy. Mechanistically, bioinformatics analysis revealed that IL-6/STAT3 signaling was significantly enriched in high DCLK1-expressing patients, and our results further revealed that DCLK1 enhanced IL-6 expression and STAT3 activation in TNBC cells, which finally gave rise to upregulated CSC traits and suppressed CD8+ T-cell activity. Inhibiting IL-6/STAT3 pathway by IL-6R antagonist, Tocilizumab or STAT3 inhibitor, S31-201 could abolish DCLK1-promoted malignant phenotypes of TNBC cells. Finally, DCLK1 was identified to be specifically and highly expressed in the mesenchymal-like subtype of TNBC and targeting DCLK1 could improve chemotherapy efficacy and activate antitumor immunity. Overall, our study revealed the potential clinical benefits of targeting DCLK1 in TNBC treatment.

Human gingival mesenchymal stem cells improve movement disorders and tyrosine hydroxylase neuronal damage in Parkinson disease rats.

BACKGROUND AIMS: Gingival mesenchymal stem cells (GMSCs) demonstrate high proliferation, trilineage differentiation and immunomodulatory properties. Parkinson disease (PD) is the second most common type of neurodegenerative disease. This study aimed to explore the effect and mechanism of GMSC-based therapy in 6-hydroxydopamine-induced PD rats. METHODS: RNA sequencing and quantitative proteomics technology was used to validate the neuroprotective role of GMSCs therapeutic in 6-Hydroxydopamine -induced PD model in vitro and in vivo. Western blotting, immunofluorescence and real-time quantitative PCR verified the molecular mechanism of GMSCs treatment. RESULTS: Intravenous injection of GMSCs improved rotation and forelimb misalignment behavior, enhanced the anti-apoptotic B-cell lymphoma 2/B-cell lymphoma 2-associated X axis, protected tyrosine hydroxylase neurons, decreased the activation of astrocytes and reduced the astrocyte marker glial fibrillary acidic protein and microglia marker ionized calcium-binding adaptor molecule 1 in the substantia nigra and striatum of PD rats. The authors found that GMSCs upregulated nerve regeneration-related molecules and inhibited metabolic disorders and the activation of signal transducer and activator of transcription 3. GMSCs showed a strong ability to protect neurons and reduce mitochondrial membrane potential damage and reactive oxygen species accumulation. The safety of GMSC transplantation was confirmed by the lack of tumor formation following subcutaneous transplantation into nude mice for up to 8 weeks. CONCLUSIONS: The authors' research helps to explain the mechanism of GMSC-based therapeutic strategies and promote potential clinical application in Parkinson disease.

8-Hydroxyquinoline a natural chelating agent from Streptomyces spp. inhibits A549 lung cancer cell lines via BCL2/STAT3 regulating pathways.

Biomolecules from Streptomyces spp. are emerging sources of natural drugs and have been focused on over the decade. The discovery of bioactive chemotherapeutic molecules from soil Streptomyces spp. has opened the medium for the search for natural drugs. In the current study, 8-HOQ was extracted and purified from soil Streptomyces spp. and was evaluated on A549 and BEAS cell lines. The apoptotic and caspase mediated pathways were evaluated using cell proliferation, dual fluorescent staining, migration, invasion and mRNA as well as protein quantification of apoptotic markers. In vitro cytotoxicity test revealed that 8-HOQ possesses potent cytotoxicity activities with IC50 values of 26 µM, 5 µM, 7.2 µM at 24 h, 48 h, and 72 h respectively against A549 lung cancer cell lines. The result also demonstrated that 8-HOQ from Streptomyces spp significantly inhibited the A549 lung cancer cell lines and activated the intrinsic pathways of apoptosis. The caspase-3 and caspase-8 activities were potentially elevated in 8-HOQ treated A549 cell lines and confirmed that 8-HOQ mediated A549 cancer cell death through the intrinsic pathway. The results explored caspase-mediated apoptosis as a mechanism underlying the inhibition of cancer cell viability in a dose-dependent manner. The expression of P53, BCL2 and STAT3 were inhibited in A549 cell lines and confirmed the metastasis inhibitory potential of 8-HOQ by blocking migration and invasion in A549 cell lines. These results indicated that 8-HOQ from Streptomyces spp. potentially inhibited growth and migration of A549 lung cancer cell lines.

A first-in-human Phase I trial of the oral p-STAT3 inhibitor WP1066 in patients with recurrent malignant glioma.

Aim: To ascertain the maximum tolerated dose (MTD)/maximum feasible dose (MFD) of WP1066 and p-STAT3 target engagement within recurrent glioblastoma (GBM) patients. Patients & methods: In a first-in-human open-label, single-center, single-arm 3 + 3 design Phase I clinical trial, eight patients were treated with WP1066 until disease progression or unacceptable toxicities. Results: In the absence of significant toxicity, the MFD was identified to be 8 mg/kg. The most common adverse event was grade 1 nausea and diarrhea in 50% of patients. No treatment-related deaths occurred; 6 of 8 patients died from disease progression and one was lost to follow-up. Of 8 patients with radiographic follow-up, all had progressive disease. The longest response duration exceeded 3.25 months. The median progression-free survival (PFS) time was 2.3 months (95% CI: 1.7 months-NA months), and 6-month PFS (PFS6) rate was 0%. The median overall survival (OS) rate was 25 months (95% CI: 22.5 months-NA months), with an estimated 1-year OS rate of 100%. Pharmacokinetic (PK) data demonstrated that at 8 mg/kg, the T1/2 was 2-3 h with a dose dependent increase in the Cmax. Immune monitoring of the peripheral blood demonstrated that there was p-STAT3 suppression starting at a dose of 1 mg/kg. Conclusion: Immune analyses indicated that WP1066 inhibited systemic immune p-STAT3. WP1066 had an MFD identified at 8 mg/kg which is the target allometric dose based on prior preclinical modeling in combination with radiation therapy and a Phase II study is being planned for newly diagnosed MGMT promoter unmethylated glioblastoma patients.

APE1 shRNA-loaded cancer stem cell-derived extracellular vesicles reverse Erlotinib resistance in non-small cell lung cancer via the IL-6/STAT3 signalling.

OBJECTIVE: Apurinic endonuclease 1 (APE1) has been suggested as an oncogene of lung tumours and our bioinformatics analysis identified the association between Erlotinib resistance and interleukin-6 (IL-6). Thus, we performed this work to delineate the mechanistic actions of APE1/IL-6 signalling in Erlotinib resistance of non-small cell lung cancer (NSCLC). METHODS: We selected human NSCLC cell lines HCC827 and PC9 to establish Erlotinib-resistant HCC827R and PC9R cells. Cancer stem cells (CSCs) were isolated from Erlotinib-sensitive HCC827P and PC9P cells (PCSCs) and from HCC827R and PC9R cells (RCSCs). Further, extracellular vesicles (EVs) were separated from PCSCs (PCSC-EVs) and RCSCs (RCSC-EVs) and co-cultured with RCSCs with or without short hairpin RNA (shRNA)-targeting APE1 (APE1 shRNA) transduction. In addition, functional assays were conducted to determine the effect of APE1 shRNA on malignant phenotypes of cancer cells in vitro and in vivo and the activation of IL-6/STAT3 signalling. RESULTS: It was found that NSCLC cells could internalize both RCSC-EVs and PCSC-EVs. RCSC-EVs augmented the resistance of NSCLC cells to Erlotinib. The overexpression of APE1 occurred in NSCLC tissues, and IL-6 was enriched in serum samples of patients with NSCLC. APE1 shRNA was demonstrated to restrict the Erlotinib resistance of NSCLC cells by inactivating the IL-6/STAT3 signalling. Additionally, shAPE1-loaded RCSC-EVs suppressed the Erlotinib resistance of NSCLC via the IL-6/STAT3 axis both in vitro and in vivo, as reflected by impeded malignant phenotypes and xenograft tumour formation. CONCLUSIONS: Collectively, these data indicate that APE1 confers Erlotinib resistance by activating the IL-6/STAT3 signalling, suggesting targeting APE1 as a possible therapeutic target in Erlotinib-resistant NSCLC.

Persistent activation of signal transducer and activator of transcription 3 via interleukin-6 trans-signaling is involved in fibrosis of endometriosis.

STUDY QUESTION: Is activation of signal transducer and activator of transcription 3 (STAT3) via interleukin-6 (IL-6) trans-signaling involved in fibrosis of endometriosis? SUMMARY ANSWER: Persistent activation of STAT3 via IL-6 trans-signaling is involved in fibrosis of endometriosis. WHAT IS KNOWN ALREADY: Our previous study showed that sustained low-grade inflammation promotes a fibrotic phenotype in endometriotic stromal cells. However, the underlying mechanisms of the establishment of non-resolving, low-grade inflammation in endometriosis remain to be clarified. STUDY DESIGN, SIZE, DURATION: Endometrial and/or endometriotic samples of 60 patients who had histological evidence of deep endometriosis and endometrial samples from 32 healthy fertile women were analyzed. PARTICIPANTS/MATERIALS, SETTING, METHODS: The effects of priming with ligands of Toll-like receptors (TLRs) 2, 3 and 4 on secretion of inflammatory mediators (tumor necrosis factor-α, C-X-C motif chemokine ligand-10 [CXCL-10], IL6 and IL-10) after a second challenge with TLR ligands in endometrial and endometriotic stromal cells were investigated. Then, the effects of IL-6/soluble (s) IL-6 receptor (R)/STAT3 signaling, as well as inhibition of STAT3 activation by knockdown of STAT3 or pharmacological inhibition (S3I-201), on the pro-fibrotic phenotype in endometrial and endometriotic stromal cells in vitro were investigated. MAIN RESULTS AND THE ROLE OF CHANCE: Priming with TLR ligands for 4 h had no significant effects, whereas 24 h of priming significantly decreased secretion of IL-6, after a second challenge in endometrial stromal cells of healthy women. In endometriotic stromal cells, whereas 24 h of priming had no significant effects, priming with TLR ligands for 4 h significantly increased secretion of IL-6 after a second challenge. IL-6/soluble IL-6 receptor (sIL-6R) induced a pro-fibrotic phenotype (cell proliferation, collagen type I synthesis, α-smooth muscle actin positive stress fibers, cell migration and collagen gel contraction) as well as nuclear factor-kappa B (NF-κB) activation of endometriotic stromal cells. In contrast, IL-6/sIL-6R had no significant effects on either a pro-fibrotic phenotype or NF-κB activation of endometrial stromal cells of healthy women. Stimulation with transforming growth factor (TGF)-ß1 and/or IL-6/sIL-6R for 1 h and 48 h activated STAT3, but induced very low or no suppressor of cytokine signaling (SOCS) 1 and 3 protein expression in endometriotic stromal cells. In endometrial stromal cells of healthy women, IL-6/sIL-6R-induced STAT3 and SOCS1/3 expression at 1 h, whereas no STAT3 activation was detected at 48 h. Knockdown of STAT3 gene or S3I-201 (a STAT3 inhibitor) decreased the IL-6/sIL-6R-induced pro-fibrotic phenotype as well as NF-κB activation and TGF-ß1-induced cell proliferation of endometriotic stromal cells. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: In vivo studies are required to confirm the present in vitro results. However, it remains challenging to mimic non-resolving chronic inflammation in animal models, as active inflammation can resolve spontaneously. WIDER IMPLICATIONS OF THE FINDINGS: Dysfunction of negative regulators of IL-6/sIL-6R/STAT3 signaling may cause persistent activation of STAT3 in endometriosis. Since STAT3 activation in the endometrium is essential for successful embryo implantation, treatment with STAT3 inhibitors would not be appropriate for women wishing to conceive. However, targeting impaired negative regulation of IL-6/sIL-6R/STAT3 signaling may still represent a promising avenue for the treatment of endometriosis. STUDY FUNDING/COMPETING INTEREST(S): This study was supported in part by the KARL STORZ SE & Co. KG (Tuttlingen, Germany). There are no conflicts of interest.

Metformin improves renal injury of MRL/lpr lupus-prone mice via the AMPK/STAT3 pathway.

OBJECTIVE: Lupus nephritis (LN) is a major complication and cause of death among patients with SLE. This research used in vivo and in vitro experiments to explore the therapeutic potential of metformin in kidney injury from LN-induced inflammation. METHODS: In vivo study, 8-week-old MRL/MpJ-Faslpr/J (MRL/lpr) mice were randomly divided into two groups (n=12 each): daily administration of 0.3 mg/mL metformin in drinking water and control (water only). Body weight and urinary samples were measured biweekly. Mice were sacrificed after 8-week treatment to harvest serum, lymph nodes, spleen and kidneys. In vitro study, human kidney-2 (HK-2) cells were pretreated with 1 mM metformin for 1 hour and then stimulated with 20 µg/mL lipopolysaccharides (LPS) or 10 ng/mL tumour necrosis factor-α (TNF-α) for another 48 hours. Protein was collected for subsequent analysis. RESULTS: We found that metformin administration improved renal function in MRL/lpr lupus-prone mice, measured by decreased urea nitrogen and urinary proteins. Metformin reduced immunoglobulin G and complement C3 deposition in glomeruli. The treatment also downregulated systemic and renal inflammation, as seen in decreased renal infiltration of F4/80-positive macrophages and reduced splenic and renal MCP-1 (monocyte chemoattractant protein-1) and TNF-α, and renal IL-1ß (interleukin 1ß) expression. Metformin administration decreased renal expression of necroptosis markers p-RIPK1 (phosphorylated receptor-interacting protein kinase 1) and p-MLKL, along with tubular injury marker KIM-1 (kidney injury molecule-1) in lupus mice. In addition, metformin alleviated the necroptosis of HK-2 cells stimulated by LPS and TNF-α, evidencing by a decrease in the expression of necroptosis markers p-RIPK1, p-RIPK3 and p-MLKL, and the inflammasome-related markers NLRP3 (NLR family pyrin domain containing 3), ASC (apoptosis-associated speck-like protein containing a CARD), caspase-1. Mechanistically, metformin treatment upregulated p-AMPK (phosphorylated AMP-activated protein kinase) and downregulated p-STAT3 (phosphorylated signal transducer and activator of transcription 3) expression in the kidneys. Moreover, AMPKα2 knockdown abolished the protective effects of metformin in vitro. CONCLUSIONS: Metformin alleviated kidney injury in LN though suppressing renal necroptosis and inflammation via the AMPK/STAT3 pathway.

Cryptotanshinone ameliorates MPP+-induced oxidative stress and apoptosis of SH-SY5Y neuroblastoma cells: the role of STAT3 in Parkinson's disease.

Cryptotanshinone (CTN) has shown its neuroprotective and anti-inflammatory qualities in non-genetic mouse model of Alzheimer's disease. According to bioinformatics analysis, CTN and Signal Transducer and Activator of Transcription 3 (STAT3) may interact to form a drug-target network. This study was conducted to identify the role of CTN-STAT3 interaction in Parkinson's disease (PD). PD model was established with MMP+-stimulated SH-SY5Y cells. After pre-treatment with CTN or co-treatment with CTN and STAT3 agonist, MTT assay was performed to observe cell viability; ELISA kit was used to measure the expression level of pro-inflammatory cytokines; DCFH-DA and corresponding assay kits were employed to determine the production of ROS, SOD, CAT and GSH-px; TUNEL assay and western blot were performed to detect cell apoptosis. STAT3 activity was also detected by western blot. Treatment with CTN alone had no impact on SH-SY5Y cell viability, but CTN pre-treatment effectively improved MPP+-induced loss of viability in SH-SY5Y cells. Moreover, pre-treatment with CTN inhibited MPP+-induced oxidative stress, apoptosis and STAT3 activity in SH-SY5Y cells, whereas this inhibitory effect was diminished after additional treatment with STAT3 agonist. CTN ameliorates MPP+-induced oxidative stress and apoptosis of SH-SY5Y neuroblastoma cells by inhibiting the expression of STAT3. Therefore, CTN could be a promising therapeutic agent, and STAT3 could be a potential target for PD treatment.

Antidepressant effects of Enterococcus faecalis 2001 through the regulation of prefrontal cortical myelination via the enhancement of CREB/BDNF and NF-κB p65/LIF/STAT3 pathways in olfactory bulbectomized mice.

A therapeutic strategy through the gut-brain axis has been proven to be effective in treatment for depression. In our previous study, we demonstrated that Enterococcus faecalis 2001 (EF-2001) prevents colitis-induced depressive-like behavior through the gut-brain axis in mice. More recently, we found that demyelination in the prefrontal cortex (PFC) was associated with depressive-like behavior in an animal model of major depressive disorder, olfactory bulbectomized (OBX) mice. The present study investigated the effects of EF-2001 on depressive-like behaviors in OBX mice and the underlying molecular mechanisms from the perspective of myelination in the PFC. OBX mice exhibited depressive-like behaviors in the tail-suspension, splash, and sucrose preference tests, and decreased myelin and paranodal proteins along with mature oligodendrocytes in the PFC. These behavioral and biochemical changes were all prevented by treatment with EF-2001. Further, EF-2001 treatment increased brain-derived neurotrophic factor (BDNF) and leukemia inhibitory factor (LIF) in the PFC. Interestingly, an immunohistochemical analysis revealed enhanced phospho (p) -cAMP-responsive element binding protein (CREB) expression in neurons, p-nuclear factor-kappa B (NFκB) p65 (Ser536) expression in astrocytes, and p-signal transducer and activator of transcription 3 (STAT3) (Ty705) expression in mature oligodendrocytes in the PFC of OBX mice. From these results, we suggest that EF-2001 administration prevents depressive-like behaviors by regulating prefrontal cortical myelination via the enhancement of CREB/BDNF and NFκB p65/LIF/STAT3 pathways. Our findings strongly support the idea that a therapeutic strategy involving the gut microbiota may be a promising alternative treatment for alleviating symptoms of depression.

Delivery of siRNA based on engineered exosomes for glioblastoma therapy by targeting STAT3.

Small interfering RNA (siRNA) therapy has been considered as a promising strategy for treatment of glioblastoma (GBM), which is an aggressive brain disease with poor prognosis. However, siRNA therapy for GBM is seriously hindered by a multitude of barriers including possible immunogenicity, poor cellular uptake, short blood circulation, poor blood stability and low blood-brain barrier (BBB) penetration. This paper reports Angiopep-2 (An2)-functionalized signal transducers and activators of transcription 3 (STAT3) siRNA-loaded exosomes (Exo-An2-siRNA) as potential therapeutic agents to improve GBM therapy. The experimental results indicate that Exo-An2-siRNA displays high blood stability, efficient cellular uptake, and outstanding BBB penetration ability. Exo-An2-siRNA also exhibits excellent in vitro anti-GBM therapeutic effects due to the exosomes for siRNA protection and An2 modification for GBM targeting and BBB penetration. Such superior properties of Exo-An2-siRNA are responsible for favorable inhibition of the proliferation of orthotopic U87MG xenografts with limited side effects, significantly enhancing the median survival time (MST) of U87MG-bearing nude mice. The developed siRNA therapy featuring An2-functionalized exosomes as nanoplatforms is a safe and effective GBM treatment strategy.

Neobavaisoflavone Demonstrates Valid Anti-tumor Effects in Non-Small- Cell Lung Cancer by Inhibiting STAT3.

AIM AND OBJECTIVE: Lung cancer is the most commonly occurring cancer, which contributes to the majority of death caused by cancer, where non-small-cell lung cancer (NSCLC) accounts for approximately 85% of lung cancer. To treat NSCLC, STAT3 has been identified as a target with therapeutic potential. The neobavaisoflavone (NBIF) is one of the flavonoids of traditional Chinese medicine Psoralea corylifolial. MATERIALS AND METHODS: Human NSCLC cell lines, PC-9, H460, and A549, were applied to determine NBIF's anti-proliferative effects through cell viability and colony formation detection. The effect of NBIF on cell apoptosis was determined through flow cytometry-based assay. Western blotting was used in this study to confirm the levels of P-STAT3, Bcl-2, and Bax, which are apoptotic proteins. RESULTS: It was observed that NBIF could decrease the cell viability and its migration and induce apoptosis in human NSCLC cell lines dose-dependently. Levels of P-STAT3, as well as the downstream signals of the STAT3 pathway, were downregulated, suggesting that the tumorsuppression effects of NBIF might be related to the inhibition of STAT3 signaling. Furthermore, NBIF could contribute to the upregulation of BAX and downregulation of BCL2. CONCLUSION: NBIF might perform the anti-NSCLC efficacy as a result of the inhibition of the STAT3 pathway. Besides, our work suggests that NBIF could provide therapeutic alternatives for NSCLC.

Co-delivery of paclitaxel and STAT3 siRNA by a multifunctional nanocomplex for targeted treatment of metastatic breast cancer.

Metastasis is one of the major causes of mortality in patients suffering from breast cancer. The signal transducer and activator of transcription 3 (STAT3) is closely related to cancer metastasis. Herein, a multifunctional nanocomplex was developed to simultaneously deliver paclitaxel (PTX) and STAT3 siRNA (siSTAT3) to inhibit tumor growth and prevent metastasis of breast cancer cells. PTX was encapsulated into the synthesized polyethyleneimine-polylactic acid-lipoic acid (PPL) micelle through hydrophobic interaction, while siSTAT3 was condensed onto polyethyleneimine through electrostatic interaction. The surface charge of the drug-loaded nanocomplex (siSTAT3PPLPTX) was then converted to negative by coating with hyaluronic acid (HA). The multifunctional nanocomplex (HA/siSTAT3PPLPTX) effectively entered CD44-overexpressed 4T1 cells via an active targeting mechanism. HA shell was degraded by the concentrated hyaluronidase in the endo/lysosome and the rapid drug release was triggered by the redox micro-environment of cytoplasm. Moreover, HA/siSTAT3PPLPTX showed enhanced cytotoxicity against tumor cells due to a synergistic effect of PTX and siSTAT3. The effective inhibition of tumor metastasis was confirmed by in vitro cell migration and invasion in 4T1 cells. More importantly, a superior antitumor efficacy was observed in orthotopic 4T1 tumor-bearing mice, with no side effects in major organs, and the lung metastasis was strongly inhibited in 4T1 metastasis model. In conclusion, the multifunctional nanocomplex provides a versatile platform for efficient treatment of metastatic cancer through tumor-targeted chemo-gene combined therapy. STATEMENT OF SIGNIFICANCE: Metastasis is one of the major causes of mortality in patients suffering from breast cancer. The signal transducer and activator of transcription 3 (STAT3) is closely related to cancer metastasis. In this study, a multifunctional nanocomplex co-loaded with paclitaxel (PTX) and STAT3 siRNA was constructed and characterized. The co-delivery system exhibited active tumor targeting, effective endo/lysosomal escape, and rapid intracellular drug release. Both in vitro and in vivo studies indicated that the nanocomplex could lead to superior tumor growth inhibition, as well as metastasis suppression by silencing expression of STAT3 and p-STAT3. This present study implies that the nanocomplex could be a potential platform for targeted treatment of metastatic cancer through chemo-gene combined therapy.

STAT3 Inhibitor ODZ10117 Suppresses Glioblastoma Malignancy and Prolongs Survival in a Glioblastoma Xenograft Model.

Constitutively activated STAT3 plays an essential role in the initiation, progression, maintenance, malignancy, and drug resistance of cancer, including glioblastoma, suggesting that STAT3 is a potential therapeutic target for cancer therapy. We recently identified ODZ10117 as a small molecule inhibitor of STAT3 and suggested that it may have an effective therapeutic utility for the STAT3-targeted cancer therapy. Here, we demonstrated the therapeutic efficacy of ODZ10117 in glioblastoma by targeting STAT3. ODZ10117 inhibited migration and invasion and induced apoptotic cell death by targeting STAT3 in glioblastoma cells and patient-derived primary glioblastoma cells. In addition, ODZ10117 suppressed stem cell properties in glioma stem cells (GSCs). Finally, the administration of ODZ10117 showed significant therapeutic efficacy in mouse xenograft models of GSCs and glioblastoma cells. Collectively, ODZ10117 is a promising therapeutic candidate for glioblastoma by targeting STAT3.

Signal transducer and activator of transcription 3 downregulation in J774A.1 cell line as a model of M2 macrophages in tumor microenvironment.

AIM: Tumor-associated macrophages (TAMs) play a decisive role in the regulation of tumor progression by manipulating tumor oncogenesis, angiogenesis, and immune functions within tumor microenvironments. Tumor progression is frequently associated with a phenotypic switch from M1 to M2 in TAM. Activation of signal transducer and activator of transcription 3 (STAT3) in TAM lead to tumor-induced immunosuppression. STAT3 is usually constitutively activated in a variety of malignancies. Consequently, STAT3 has emerged as a promising target for cancer immunotherapy. MATERIALS AND METHODS: In this study, J774A.1 cell line which is an M2 macrophage and overexpress STAT3 was cultured in Dulbecco's Modified Eagle Medium supplemented by fetal bovine serum. Then, the STAT3 silencing was evaluated by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) using oligofectamine containing STAT3 short interfering RNA (siRNA). Oligofectamine containing STAT3 siRNA and control siRNA were added at a final concentration of 100 nM siRNA. The untransfected cells were considered as control group. RESULTS: The semi-quantitative RT-PCR studies showed that J774A.1 cells express a high level of STAT3. Incubation of J774A.1 cells with oligofectamine containing STAT3 siRNA knockdown the STAT3 expression significantly both in 48 and 72 h study; however, the effect was more pronounced in 72 h study. CONCLUSION: The expression of STAT3 in J774A.1 cells confirmed that these cells are M2 macrophage. Moreover, silencing of STAT3 by siRNA delivery using oligofectamine delivery suggests that siRNA delivery using vehicles like nanoliposome could be a useful therapeutic agent in M2 macrophage therapy and its switch to M1 macrophages. This approach could be considered as a novel therapeutic agent for the treatment of all cancers.

A First-in-Human Phase I Study of OPB-111077, a Small-Molecule STAT3 and Oxidative Phosphorylation Inhibitor, in Patients with Advanced Cancers.

LESSONS LEARNED: OPB-111077 is a novel inhibitor of STAT3 and mitochondrial oxidative phosphorylation that exhibited promising anticancer activity in preclinical models.In this first-in-human phase I study of OPB-111077 in unselected advanced cancers, treatment-emergent adverse events, most frequently nausea, fatigue, and vomiting, were generally mild to moderate in intensity and could be medically managed.Overall, only modest clinical activity was observed after OPB-111077 given as monotherapy. Notable antitumor activity was seen in a subject with diffuse large B-cell lymphoma. BACKGROUND: OPB-111077 is a novel inhibitor of STAT3 and mitochondrial oxidative phosphorylation with promising anticancer activity in preclinical models. METHODS: Open-label, phase I trial of OPB-111077 in advanced cancers with no available therapy of documented benefit. Initial dose escalation in unselected subjects was followed by dose expansion. Patients received oral OPB-111077 daily in 28-day cycles until loss of clinical benefit. RESULTS: Eighteen subjects enrolled in dose escalation, and 127 in dose expansion. Dose-limiting toxicities were observed at 300 mg and 400 mg QD; maximum tolerated dose was defined as 250 mg QD. Frequently reported treatment-emergent adverse events (TEAEs) included nausea, fatigue, and vomiting. TEAEs were generally mild to moderate and could be medically managed. OPB-111077 reached micromolar drug concentrations, had an elimination half-life of approximately 1 day, and reached steady-state by day 8. A durable partial response was observed in one subject with diffuse large B-cell lymphoma. Seven subjects with diverse tumor types had stable disease or minor responses for at least eight treatment cycles (224 days). CONCLUSION: OPB-111077 is generally well tolerated, and its pharmacokinetic profile is sufficient for further clinical development. Notable clinical activity was observed in a subject with diffuse large B-cell lymphoma. Overall, modest efficacy was observed against unselected tumors.

A novel chimeric antigen receptor containing a JAK-STAT signaling domain mediates superior antitumor effects.

The adoptive transfer of T cells engineered with a chimeric antigen receptor (CAR) (hereafter referred to as CAR-T cells) specific for the B lymphocyte antigen CD19 has shown impressive clinical responses in patients with refractory B cell malignancies. However, the therapeutic effects of CAR-T cells that target other malignancies have not yet resulted in significant clinical benefit. Although inefficient tumor trafficking and various immunosuppressive mechanisms can impede CAR-T cell effector responses, the signals delivered by the current CAR constructs may still be insufficient to fully activate antitumor T cell functions. Optimal T cell activation and proliferation requires multiple signals, including T cell receptor (TCR) engagement (signal 1), co-stimulation (signal 2) and cytokine engagement (signal 3). However, CAR constructs currently being tested in the clinic contain a CD3z (TCR signaling) domain and co-stimulatory domain(s) but not a domain that transmits signal 3 (refs. 13, 14, 15, 16, 17, 18). Here we have developed a novel CAR construct capable of inducing cytokine signaling after antigen stimulation. This new-generation CD19 CAR encodes a truncated cytoplasmic domain from the interleukin (IL)-2 receptor ß-chain (IL-2Rß) and a STAT3-binding tyrosine-X-X-glutamine (YXXQ) motif, together with the TCR signaling (CD3z) and co-stimulatory (CD28) domains (hereafter referred to as 28-ΔIL2RB-z(YXXQ)). The 28-ΔIL2RB-z(YXXQ) CAR-T cells showed antigen-dependent activation of the JAK kinase and of the STAT3 and STAT5 transcription factors signaling pathways, which promoted their proliferation and prevented terminal differentiation in vitro. The 28-ΔIL2RB-z(YXXQ) CAR-T cells demonstrated superior in vivo persistence and antitumor effects in models of liquid and solid tumors as compared with CAR-T cells expressing a CD28 or 4-1BB co-stimulatory domain alone. Taken together, these results suggest that our new-generation CAR has the potential to demonstrate superior antitumor effects with minimal toxicity in the clinic and that clinical translation of this novel CAR is warranted.