Evodiamine Exhibits Anti-Bladder Cancer Activity by Suppression of Glutathione Peroxidase 4 and Induction of Ferroptosis.

Evodiamine (EVO) exhibits anti-cancer activity through the inhibition of cell proliferation; however, little is known about its underlying mechanism. To determine whether ferroptosis is involved in the therapeutic effects of EVO, we investigated critical factors, such as lipid peroxidation levels and glutathione peroxidase 4 (GPX4) expression, under EVO treatment. Our results showed that EVO inhibited the cell proliferation of poorly differentiated, high-grade bladder cancer TCCSUP cells in a dose- and time-dependent manner. Lipid peroxides were detected by fluorescence microscopy after cancer cell exposure to EVO. GPX4, which catalyzes the conversion of lipid peroxides to prevent cells from undergoing ferroptosis, was decreased dose-dependently by EVO treatment. Given the features of iron dependency and lipid-peroxidation-driven death in ferroptosis, the iron chelator deferoxamine (DFO) was used to suppress EVO-induced ferroptosis. The lipid peroxide level significantly decreased when cells were treated with DFO prior to EVO treatment. DFO also attenuated EVO-induced cell death. Co-treatment with a pan-caspase inhibitor or necroptosis inhibitor with EVO did not alleviate cancer cell death. These results indicate that EVO induces ferroptosis rather than apoptosis or necroptosis. Furthermore, EVO suppressed the migratory ability, decreased the expression of mesenchymal markers, and increased epithelial marker expression, determined by a transwell migration assay and Western blotting. The TCCSUP bladder tumor xenograft tumor model confirmed the effects of EVO on the inhibition of tumor growth and EMT. In conclusion, EVO is a novel inducer for activating the ferroptosis of bladder cancer cells and may be a potential therapeutic agent for bladder cancer.

Interruption of the long non-coding RNA HOTAIR signaling axis ameliorates chemotherapy-induced cachexia in bladder cancer.

BACKGROUND: Cisplatin-based chemotherapy is the first line of treatment for bladder cancer. However, cisplatin induces muscle wasting associated with NF-κB and cancer cachexia. HOTAIR, an oncogenic long non-coding RNA (lncRNA), promotes cancer progression in different cancers. Crosstalk between HOTAIR and NF-κB is documented. Prothymosin α (ProT) plays important roles in cancer progression and inflammation. However, the potential link between HOTAIR, ProT, and cisplatin-induced cancer cachexia remains unexplored. Here, we investigated the contribution of HOTAIR in cisplatin-induced cancer cachexia and dissected the potential signaling cascade involving the epidermal growth factor receptor (EGFR), ProT, NF-κB, and HOTAIR. MATERIALS AND METHODS: Expression of ProT and HOTAIR transcripts and their correlations in tumor tissues of bladder cancer patients and bladder cancer cell lines were determined by RT-qPCR. Next, levels of phospho-EGFR, EGFR, phospho-NF-κB, and NF-κB were examined by immunoblot analysis in human bladder cancer cells treated with cisplatin. Expression of HOTAIR in cisplatin-treated cells was also assessed by RT-qPCR. Pharmacological inhibitors and overexpression and knockdown approaches were exploited to decipher the signaling pathway. The murine C2C12 myoblasts were used as an in vitro muscle atrophy model. The syngeneic murine MBT-2 bladder tumor was used to investigate the role of mouse Hotair in cisplatin-induced cancer cachexia. RESULTS: Expression of ProT and HOTAIR was higher in bladder tumors than in normal adjacent tissues. There were positive correlations between ProT and HOTAIR expression in clinical bladder tumors and bladder cancer cell lines. Cisplatin treatment increased EGFR and NF-κB activation and upregulated ProT and HOTAIR expression in bladder cancer cells. ProT overexpression increased, whereas ProT knockdown decreased, HOTAIR expression. Notably, cisplatin-induced HOTAIR upregulation was abrogated by EGFR inhibitors or ProT knockdown. ProT-induced HOTAIR overexpression was diminished by NF-κB inhibitors. HOTAIR overexpression enhanced, whereas its knockdown reduced, cell proliferation, cachexia-associated pro-inflammatory cytokine expression, and muscle atrophy. Cachexia-associated symptoms were ameliorated in mice bearing Hotair-knockdown bladder tumors undergoing cisplatin treatment. CONCLUSIONS: We demonstrate for the first time a critical role for HOTAIR and identify the involvement of the EGFR-ProT-NF-κB-HOTAIR signaling axis in cisplatin-induced cachexia in bladder cancer and likely other cancers. Our findings also provide therapeutic targets for this disease.

Prothymosin α promotes STAT3 acetylation to induce cystogenesis in Pkd1-deficient mice.

Polycystic kidney disease (PKD) is characterized by the expansion of fluid-filled cysts in the kidney, which impair the function of kidney and eventually leads to end-stage renal failure. It has been previously demonstrated that transgenic overexpression of prothymosin α (ProT) induces the development of PKD; however, the underlying mechanisms remain unclear. In this study, we used a mouse PKD model that sustains kidney-specific low-expression of Pkd1 to illustrate that aberrant up-regulation of ProT occurs in cyst-lining epithelial cells, and we further developed an in vitro cystogenesis model to demonstrate that the suppression of ProT is sufficient to reduce cyst formation. Next, we found that the expression of ProT was accompanied with prominent augmentation of protein acetylation in PKD, which results in the activation of downstream signal transducer and activator of transcription (STAT) 3. The pathologic role of STAT3 in PKD has been previously reported. We determined that this molecular mechanism of protein acetylation is involved with the interaction between ProT and STAT3; consequently, it causes the deprivation of histone deacetylase 3 from the indicated protein. Conclusively, these results elucidate the significant role of ProT, including protein acetylation and STAT3 activation in PKD, which represent potential for ameliorating the disease progression of PKD.-Chen, Y.-C., Su, Y.-C., Shieh, G.-S., Su, B.-H., Su, W.-C., Huang, P.-H., Jiang, S.-T., Shiau, A.-L., Wu, C.-L. Prothymosin α promotes STAT3 acetylation to induce cystogenesis in Pkd1-deficient mice.

Oct4 upregulates osteopontin via Egr1 and is associated with poor outcome in human lung cancer.

BACKGROUND: Roles of cancer stem cells and early growth response gene 1 (Egr1) in carcinogenesis have been extensively studied in lung cancer. However, the role of Egr1 in the metastasis of lung cancer remains undetermined, especially in regard to stem cell-related pathways. METHODS: Egr1, osteopontin (OPN) and Oct4 expression in human lung cancer was determined by performing immunohistochemistry. Immunoblotting, ELISA, luciferase reporter assay, chromatin immunoprecipitation assay and RT-PCR were performed to validate the regulation of Oct4-Egr1-OPN axis. Moreover, the effect of Oct4-Egr1-OPN axis on lung cancer progression was evaluated by cell migration assay and mice study. RESULTS: We detected Oct4, Egr1, and OPN expression in clinical specimens from 79 lung cancer patients, including 72 adenocarcinomas and 7 squamous cell carcinomas. High expression of Oct4, Egr1, and OPN accounted for 53, 51, and 57% of the patients, respectively. All of the three biomarkers were positively correlated in clinical human lung cancer. Patients with high expression of OPN were significantly associated with shorter disease-free survivals than those with low expression of OPN (p < 0.05). In lung cancer cells, Oct4 transactivated the Egr1 promoter and upregulated Egr1 expression. In a human lung cancer xenograft model, Oct4-overexpressing tumors expressed elevated levels of Egr1. Furthermore, overexpression of Oct4 in lung cancer cells increased the metastatic potential. CONCLUSIONS: Egr1 exerts a promoting effect on cancer metastasis in Oct4-overexpressing lung cancer. Thus, therapeutic strategies targeting the Oct4/Egr1/OPN axis may be further explored for the treatment of lung cancer, especially when lung cancer is refractory to conventional treatment due to cancer stem cells.

Over-expression of prothymosin-α antagonizes TGFß signalling to promote the development of emphysema.

Emphysema, a major consequence of chronic obstructive pulmonary disease (COPD), is characterized by the permanent airflow restriction resulting from enlargement of alveolar airspace and loss of lung elasticity. Transforming growth factor-ß (TGFß) signalling regulates the balance of matrix metalloproteinase (MMP)/tissue inhibitor of matrix metalloproteinase (TIMP) to control matrix homeostasis. Patients with COPD have dysregulated TGFß signalling and reduced histone deacetylase (HDAC) activity through epigenetic up-regulation of histone acetylation in the promoters of pro-inflammatory genes. However, the potential link between decreased HDAC activity and dysregulated TGFß signalling in emphysema pathogenesis remains to be determined. Prothymosin α (ProT), a highly conserved acidic nuclear protein, plays a role in the acetylation of histone and non-histone proteins. The aim of this study was to test the hypothesis that ProT inhibits TGFß-Smad signalling through Smad7, thereby contributing to emphysema pathogenesis. We show that ProT enhances Smad7 acetylation by decreasing its association with HDAC and thereby down-regulates TGFß-Smad signalling. ProT caused an imbalance between MMP and TIMP through acetylated Smad7 in favour of MMP expression. In addition to interfering with R-Smad activation and targeting receptors for degradation in the cytoplasm, acetylated Smad7 potentiated by ProT competitively antagonized binding of the pSmad2/3-Smad4 complex to the TIMP-3 promoter, resulting in reduced TIMP-3 expression. These effects were detected in ProT-over-expressing cells, lungs of ProT transgenic mice displaying an emphysema phenotype and in emphysema patients. Importantly, increased Smad7 and reduced TIMP-3 were found in the lungs of emphysema patients and mice with cigarette smoke extract (CSE)-induced emphysema. Such effects could be abrogated by silencing endogenous ProT expression. Collectively, our results uncover acetylated Smad7 regulated by ProT as an important determinant in dysregulated TGFß signalling that contributes to emphysema pathogenesis.

Kallistatin ameliorates influenza virus pathogenesis by inhibition of kallikrein-related peptidase 1-mediated cleavage of viral hemagglutinin.

Proteolytic cleavage of the hemagglutinin (HA) of influenza virus by host trypsin-like proteases is required for viral infectivity. Some serine proteases are capable of cleaving influenza virus HA, whereas some serine protease inhibitors (serpins) inhibit the HA cleavage in various cell types. Kallikrein-related peptidase 1 (KLK1, also known as tissue kallikrein) is a widely distributed serine protease. Kallistatin, a serpin synthesized mainly in the liver and rapidly secreted into the circulation, forms complexes with KLK1 and inhibits its activity. Here, we investigated the roles of KLK1 and kallistatin in influenza virus infection. We show that the levels of KLK1 increased, whereas those of kallistatin decreased, in the lungs of mice during influenza virus infection. KLK1 cleaved H1, H2, and H3 HA molecules and consequently enhanced viral production. In contrast, kallistatin inhibited KLK1-mediated HA cleavage and reduced viral production. Cells transduced with the kallistatin gene secreted kallistatin extracellularly, which rendered them more resistant to influenza virus infection. Furthermore, lentivirus-mediated kallistatin gene delivery protected mice against lethal influenza virus challenge by reducing the viral load, inflammation, and injury in the lung. Taking the data together, we determined that KLK1 and kallistatin contribute to the pathogenesis of influenza virus by affecting the cleavage of the HA peptide and inflammatory responses. This study provides a proof of principle for the potential therapeutic application of kallistatin or other KLK1 inhibitors for influenza. Since proteolytic activation also enhances the infectivity of some other viruses, kallistatin and other kallikrein inhibitors may be explored as antiviral agents against these viruses.

Syngeneic mesenchymal stem cells loaded with telomerase-dependent oncolytic adenoviruses enhance anti-metastatic efficacy.

Oncolytic adenoviruses have emerged as a promising therapeutic approach for cancer therapy. However, systemic delivery of the viruses to metastatic tumors remains a major challenge. Mesenchymal stem cells (MSCs) possess tumor tropism property and can be used as cellular vehicles for delivering oncolytic adenoviruses to tumor sites. Since telomerase activity is found in ~90% of human carcinomas, but undetected in normal adult cells, the human telomerase reverse transcriptase gene (TERT) promoter can be exploited for regulating the replication of oncolytic adenoviruses. Here, we evaluated the antitumor effects of syngeneic murine MSCs loaded with the luciferase-expressing, telomerase-dependent oncolytic adenovirus Ad.GS2 (MSC-Ad.GS2) and Ad.GS2 alone on metastatic MBT-2 bladder tumors. MSCs supported a low degree of Ad.GS2 replication, which could be augmented by coculture with MBT-2 cells or tumor-conditioned medium (TCM), suggesting that viral replication is increased when MSC-Ad.GS2 migrates to tumor sites. MBT-2 cells and TCM enhanced viral replication in Ad.GS2-infected MSCs. SDF-1 is a stem cell homing factor. Our results suggest that the SDF-1/STAT3/TERT signaling axis in MSCs in response to the tumor microenvironment may contribute to the enhanced replication of Ad.GS2 carried by MSCs. Notably, we demonstrate the potent therapeutic efficacy of systemically delivered MSC-Ad.GS2 in pleural disseminated tumor and experimental metastasis models using intrapleural and tail vein injection of MBT-2 cells, respectively. Treatment with MSC-Ad.GS2 significantly reduced tumor growth and prolonged the survival of mice bearing metastatic bladder tumors. Since telomerase is expressed in a broad spectrum of cancers, this therapeutic strategy may be broadly applicable.

Oct4 and Hypoxia Dual-Regulated Oncolytic Adenovirus Armed with shRNA-Targeting Dendritic Cell Immunoreceptor Exerts Potent Antitumor Activity against Bladder Cancer.

Immunotherapy has emerged as a promising modality for cancer treatment. Dendritic cell immunoreceptor (DCIR), a C-type lectin receptor, is expressed mainly by dendritic cells (DCs) and mediates inhibitory intracellular signaling. Inhibition of DCIR activation may enhance antitumor activity. DCIR is encoded by CLEC4A in humans and by Clec4a2 in mice. Gene gun-mediated delivery of short hairpin RNA (shRNA) targeting Clec4a2 into mice bearing bladder tumors reduces DCIR expression in DCs, inhibiting tumor growth and inducing CD8+ T cell immune responses. Various oncolytic adenoviruses have been developed in clinical trials. Previously, we have developed Ad.LCY, an oncolytic adenovirus regulated by Oct4 and hypoxia, and demonstrated its antitumor efficacy. Here, we generated a Clec4a2 shRNA-expressing oncolytic adenovirus derived from Ad.LCY, designated Ad.shDCIR, aimed at inducing more robust antitumor immune responses. Our results show that treatment with Ad.shDCIR reduced Clec4a expression in DCs in cell culture. Furthermore, Ad.shDCIR exerted cytolytic effects solely on MBT-2 bladder cancer cells but not on normal NIH 3T3 mouse fibroblasts, confirming the tumor selectivity of Ad.shDCIR. Compared to Ad.LCY, Ad.shDCIR induced higher cytotoxic T lymphocyte (CTL) activity in MBT-2 tumor-bearing immunocompetent mice. In addition, Ad.shDCIR and Ad.LCY exhibited similar antitumor effects on inhibiting tumor growth. Notably, Ad.shDCIR was superior to Ad.LCY in prolonging the survival of tumor-bearing mice. In conclusion, Ad.shDCIR may be further explored as a combination therapy of virotherapy and immunotherapy for bladder cancer and likely other types of cancer.

Galectin-1 binds to influenza virus and ameliorates influenza virus pathogenesis.

Innate immune response is important for viral clearance during influenza virus infection. Galectin-1, which belongs to S-type lectins, contains a conserved carbohydrate recognition domain that recognizes galactose-containing oligosaccharides. Since the envelope proteins of influenza virus are highly glycosylated, we studied the role of galectin-1 in influenza virus infection in vitro and in mice. We found that galectin-1 was upregulated in the lungs of mice during influenza virus infection. There was a positive correlation between galectin-1 levels and viral loads during the acute phase of viral infection. Cells treated with recombinant human galectin-1 generated lower viral yields after influenza virus infection. Galectin-1 could directly bind to the envelope glycoproteins of influenza A/WSN/33 virus and inhibit its hemagglutination activity and infectivity. It also bound to different subtypes of influenza A virus with micromolar dissociation constant (K(d)) values and protected cells against influenza virus-induced cell death. We used nanoparticle, surface plasmon resonance analysis and transmission electron microscopy to further demonstrate the direct binding of galectin-1 to influenza virus. More importantly, we show for the first time that intranasal treatment of galectin-1 could enhance survival of mice against lethal challenge with influenza virus by reducing viral load, inflammation, and apoptosis in the lung. Furthermore, galectin-1 knockout mice were more susceptible to influenza virus infection than wild-type mice. Collectively, our results indicate that galectin-1 has anti-influenza virus activity by binding to viral surface and inhibiting its infectivity. Thus, galectin-1 may be further explored as a novel therapeutic agent for influenza.

RISC-V CNN Coprocessor for Real-Time Epilepsy Detection in Wearable Application.

Epilepsy is a common clinical disease. Severe epilepsy can be life-threatening in certain unexpected conditions, so it is important to detect seizures instantly with a wearable device and to provide treatment within the golden window. The observation of the electroencephalography (EEG) signal is an imperative method to assist correct epilepsy diagnosis. To detect and classify EEG signals, a convolutional neural network (CNN) is an intuitive and appropriate method that borrows expertise from neurologists. However, the computational cost of training and inference on artificial intelligence (AI)-based solutions make software-only and hardware-only solutions incompetent for real-time monitoring on embedded devices. Hence, this study proposes three key contributions for the challenge, namely, an algorithm framework to provide real-time epilepsy detection, a dedicated coprocessor chip implementing this framework to enable real time epilepsy detection to offload and accelerate detection algorithm, and a custom interface with the coprocessor and reduced instruction set computer-V (RISC-V) instructions to reconfigure the coprocessor and transfer data. The epilepsy detection framework is implemented in 11-layer CNN. The proposed epilepsy detection algorithm performs 97.8% accuracy for floating-point and 93.5% for fixed-point operations through animal experiments with lab rats. The RISC-V CNN coprocessor is fabricated in the TSMC 0.18-µm CMOS process. For each classification, the coprocessor consumes 51 nJ/class. and 0.9 µJ/class. energy on data transfer and inference, respectively. The detection latency on the chip is 0.012 s. With the integration of the hardware coprocessor, AI algorithms can be applied to epilepsy detection for real-time monitoring.

The Pro-Survival Oct4/Stat1/Mcl-1 Axis Is Associated with Poor Prognosis in Lung Adenocarcinoma Patients.

The embryonic stem cell marker Oct4 is expressed in several human cancers and is positively correlated with a poor outcome in cancer patients. However, its physiological role in cancer progression remains poorly understood. Tumor cells block apoptosis to escape cell death so that they can proliferate indefinitely, leading to ineffective therapy for cancer patients. In this study, we investigated whether Oct4 regulates the apoptosis pathway and contributes to poor prognosis in patients with lung adenocarcinoma. Our results revealed that Oct4 expression is correlated with Stat1 expression in lung adenocarcinoma patients and Oct4 is directly bound to the Stat1 promoter to transactivate Stat1 in lung adenocarcinoma cells. Expression of the Stat1 downstream gene Mcl-1 increased in Oct4-overexpressing cancer cells, while Stat1 knockdown in Oct4-overexpressing cancer cells sensitized them to cisplatin-induced apoptosis. Furthermore, Oct4 promoted Stat1 expression and tumor growth, whereas silencing of Stat1 reduced Oct4-induced tumor growth in human lung tumor xenograft models. Taken together, we demonstrate that Oct4 is a pro-survival factor by inducing Stat1 expression and that the Oct4/Stat1/Mcl-1 axis may be a potential therapeutic target for lung adenocarcinoma.

Sprouty2 protein enhances the response to gefitinib through epidermal growth factor receptor in colon cancer cells.

Sprouty2 (Spry2) is known to increase the expression of epidermal growth factor receptors (EGFR) by conjugating with c-Casitas B-lineage lymphoma (C-Cbl) to decrease protein degradation. The effect of Spry2 on the treatment of gefitinib, a tyrosine kinase inhibitor of EGFR, with regards to colon cancer is still unclear. The half maximal inhibitory concentration (IC50) values of gefitinib in six colon cancer cell lines were assessed. HCT116 and C2BBel cells expressed lower levels of Spry2 protein and were less sensitive to gefitinib, whereas HT29 cells that expressed high levels of Spry2 protein were more sensitive to gefitinib. The sensitivity to gefitinib was increased after overexpression of Spry2 in HCT116 cells, whereas it was decreased after Spry2 knockdown in HT29 cells. The levels of both phosphorylated and total EGFR were increased when HCT116 cells ectopically overexpressed Spry2, with concomitant increase in phosphatase and tensin homolog (PTEN) expression. Inhibition of EGFR by cetuximab reduced sensitivity to gefitinib in HCT116 cells overexpressing Spry2. However, knockdown of PTEN or K-ras failed to diminish the effect of Spry2 on gefitinib sensitivity. Of note, Spry2 enhanced the antitumor effect of gefitinib in a xenograft model of HCT116 tumors, which harbored K-ras codon 13 mutation. In conclusion, Spry2 can enhance the response of colon cancer cells to gefitinib by increasing the expression of phosphorylated and total EGFR. These results suggest that Spry2 may be a potential biomarker in predicting the response to anti-EGFR treatment in colon cancer and that it is necessary to conduct clinical studies to incorporate Spry2 into the network of cancer treatment.

A 2.4 GHz ISM Band OOK Transceiver With High Energy Efficiency for Biomedical Implantable Applications.

This article presents a high energy efficiency, high-integrated, and low-power on-off keying transceiver for a 2.4 GHz industrial scientific medical band. The proposed receiver includes an input matching network, a low-noise amplifier, a novel single-to-differential envelope detector, a level shifter, cascaded baseband amplifiers, and a hysteresis comparator. The proposed transmitter includes a bias-stimulating circuit, a current-reused self-mixing voltage controlled oscillator, and a quadruple-transconductance power amplifier. Numerous proposed techniques implemented in the mentioned circuits improve the energy per bit and power efficiency. Therefore, the proposed receiver for short-distanced propagation can achieve a sensitivity of -46 dBm with a carrier frequency of 2.45 GHz and a high data rate of 2 Mbps. The proposed transmitter achieves an output power of -17 dBm with a high data rate of 20 Mbps. This work is fabricated in a TSMC 0.18 µm CMOS process and consumes 160 µW and 0.6 mW in the receiver and transmitter, respectively, from a 1.2 V supply voltage. The energy per bit of 80 pJ/bit in the receiver part and the figure of merit of 9 in the transmitter part are better than those of existing state-of-the-art transceivers.

Effects of Chinese and Western Medicine on Patients with Dengue Fever.

Dengue fever is an important epidemic disease with a high prevalence in tropical and subtropical countries. We aimed to investigate the effects of a treatment integrating traditional Chinese (TCM) and Western medicines on dengue inpatients with warning signs (i.e., group B) according to the World Health Organization dengue classification in this retrospective cohort study of medical records. Inpatients who were treated with conventional Western therapies in the absence or presence of TCM were assigned to the control and treatment groups, respectively. Data were compared using an analysis of variance, general linear analysis, and chi-square test. The most common clinical symptoms and signs of dengue fever were fever and muscle ache. The treatment group patients were significantly more likely to present general weakness and poor appetite than the control group patients. Patients in the treatment group were more likely to experience stomachache than those in the control group. Moreover, comparisons of the changes in hemoglobin and alanine aminotransferase levels over time revealed significant differences between the patient groups. Zhu Ye Shi Gao Tang, Gui Pi Tang, Paeonia suffruticosa, and Clerodendrum cyrtophyllum were the most commonly administered TCM formula and single herbs in this study. Patients in the treatment group experienced a resolution of symptoms, signs, and laboratory data and were discharged smoothly, without deterioration to death or critical care. Our findings suggest that the integration of TCM and Western medicine may yield an appropriate treatment for dengue fever.

Oct4 promotes M2 macrophage polarization through upregulation of macrophage colony-stimulating factor in lung cancer.

BACKGROUND: Expression of Oct4 maintains cancer stem cell (CSC)-like properties in lung cancer cells and is correlated with poor prognosis of lung adenocarcinoma. M2-type tumor-associated macrophages (TAMs) promote cancer cell migration and metastasis. Tumor microenvironments promote monocyte differentiation into M2 TAMs via a complex cytokine-based connection. We explored the role of Oct4 in cytokine secretion in lung cancer and its impact on M2 TAM polarization. METHODS: Monocytes co-cultured with the conditioned medium from Oct4-overexpressing lung cancer cells were used to investigate M2 TAM differentiation. The inflammatory factors in the conditioned medium of Oct4-overexpressing A549 cells were examined using human inflammation antibody arrays. The correlations of Oct4, macrophage colony-stimulating factor (M-CSF), and M2 TAMs were validated in lung cancer cells, syngeneic mouse lung tumor models, and clinical samples of non-small cell lung cancer (NSCLC). RESULTS: Oct4-overexpressing A549 cells expressed elevated levels of M-CSF, which contributed to increased M2 macrophages and enhanced tumor migration. Overexpression of Oct4 enhanced tumor growth and reduced the survival of lung tumor-bearing mice, which was correlated with increased number of M2 macrophages in lung cancer. Notably, NSCLC patients with high expression levels of Oct4, M-CSF, and M2 TAMs had the poorest recurrence-free survival. A positive correlation between Oct4, M-CSF, and M2 TAMs was observed in the tumor tissue of NSCLC patient. Treatment with all-trans retinoic acid exerted anti-tumor effects and reduced M2 TAMs in tumor-bearing mice. CONCLUSIONS: Our results indicate that Oct4 expressed by lung cancer cells promotes M2 macrophage polarization through upregulation of M-CSF secretion, leading to cancer growth and metastasis. Our findings also implicate that the Oct4/M-CSF axis in M2 macrophage polarization may be potential therapeutic targets for lung cancer.

Tumor-selective replication of an oncolytic adenovirus carrying oct-3/4 response elements in murine metastatic bladder cancer models.

PURPOSE: Oncolytic adenoviruses are attractive therapeutics for cancer because they selectively replicate in tumors. However, targeting tumor metastasis remains a major challenge for current virotherapy for cancer. Oct-3/4 is specifically expressed in embryonic stem cells and tumor cells. Oct-3/4 highly expressed in cancer cells may be a potential target for cancer therapy. We developed an E1B-55 kDa-deleted adenovirus, designated Ad.9OC, driven by nine copies of Oct-3/4 response element for treating Oct-3/4-expressing metastatic bladder cancer. EXPERIMENTAL DESIGN: We examined the expression of Oct-3/4 in human bladder tumor tissues and bladder cancer cell lines. We also evaluated the cytolytic and antitumor effects of Ad.9OC on bladder cancer cells in vitro and in vivo. RESULTS: Oct-3/4 expression was detected in bladder cancer cell lines, as well as in human bladder tumor tissues. Notably, Oct-3/4 expression was higher in metastatic compared with nonmetastatic bladder cancer cells. Ad.9OC induced higher cytolytic activity in metastatic bladder cancer cells than in their nonmetastatic counterparts, whereas it did not cause cytotoxicity in normal cells. Pharmacologic and short hairpin RNA-mediated Oct-3/4 inhibition rendered bladder cancer cells more resistant to Ad.9OC-induced cytolysis. Replication of Ad.9OC was detected in murine bladder cancer cells and bladder tumor tissues. We also showed the effectiveness of Ad.9OC for treating bladder cancer in subcutaneous, as well as metastatic, bladder tumor models. CONCLUSIONS: Ad.9OC may have therapeutic potential for treating Oct-3/4-expressing tumors. Especially, metastatic bladder tumors are good target for Ad.9OC treatment. Because Oct-3/4 is expressed in a broad spectrum of cancers, Ad.9OC may be broadly applicable.

Development of a conditionally replicating pseudorabies virus for HER-2/neu-overexpressing bladder cancer therapy.

Overexpression of the HER-2/neu oncogene, a frequent molecular event in a variety of cancers including bladder cancer, is associated with tumor progression and poor prognosis. Therapeutic strategies to targeting HER-2/neu-overexpressing cancer cells have shown promise. Pseudorabies virus (PrV), a herpesvirus of swine, may be exploited as an oncolytic agent for human cancer. Herein, we generated a conditionally replicating glycoprotein E-defective PrV mutant carrying glycoprotein D and herpes simplex virus type 1 thymidine kinase genes, which are essential for viral entry and replication, under the transcriptional control of the HER-2/neu promoter. The recombinant PrV, designated YP2, selectively replicated in and lysed HER-2/neu-overexpressing human bladder, mouse bladder, and hamster oral cancer cells in vitro. Notably, YP2 retarded MBT-2 bladder tumor growth in mice by more than 50% and more than half of the mice survived for over 50 days, whereas all the control mice survived less than 30 days. Taken together, our results suggest that YP2 may have therapeutic potential for the treatment of invasive bladder cancer. Furthermore, because HER-2/neu is overexpressed in a broad spectrum of cancers, this conditionally replicating PrV may be broadly applicable.

Low-dose etoposide enhances telomerase-dependent adenovirus-mediated cytosine deaminase gene therapy through augmentation of adenoviral infection and transgene expression in a syngeneic bladder tumor model.

The human telomerase reverse transcriptase (hTERT) promoter can selectively drive transgene expression in many telomerase-positive human cancer cells. Here we evaluated combination therapy of adenoviral vector Ad-hTERT-CD encoding E. coli cytosine deaminase (CD) driven by the hTERT promoter and low-dose etoposide (0.1 microg/mL) for treating bladder cancer. Ad-hTERT-CD conferred sensitivity to 5-fluorocytosine (5-FC) in bladder cancer cells, which could be enhanced by etoposide treatment, but not in normal cells. Such effect was correlated with up-regulation of hypoxia-inducible factor (HIF)-1alpha expression. By contrast, etoposide activated p53 and down-regulated hTERT promoter activity in normal cells. Etoposide also increased adenoviral infection via enhancement of coxsackie-adenovirus receptor expression on bladder cancer and normal cells. Combination index analysis revealed that combined therapy of Ad-hTERT-CD (10(9) plaque-forming units)/5-FC (200 mg/kg) with etoposide (2 mg/kg) synergistically suppressed tumor growth and prolonged survival in mice bearing syngeneic MBT-2 bladder tumors. This combination therapy regimen induced complete tumor regression and generated antitumor immunity in 75% of tumor-bearing mice. Furthermore, increased infiltrating CD4(+) and CD8(+) T cells and necrosis within tumors were found in mice receiving combination therapy of Ad-hTERT-CD and etoposide compared with those treated with either treatment alone. Thus, the potential high therapeutic index of the combination therapy may be an appealing therapeutic intervention for bladder cancer. Furthermore, because a majority of human tumors exhibit high telomerase activity, adenovirus-mediated CD gene therapy driven by the hTERT promoter in combination with low-dose etoposide may be applicable to a broad spectrum of cancers.

Chemotherapeutics-induced Oct4 expression contributes to drug resistance and tumor recurrence in bladder cancer.

Cancer cells initially characterized as sensitive to chemotherapy may acquire resistance to chemotherapy and lead to tumor recurrence through the expansion of drug-resistant population. Acquisition of drug resistance to conventional chemotherapy is a major obstacle in the treatment of recurrent cancer. Here we investigated whether anticancer drugs induced Oct4 expression, thereby contributing to acquired drug resistance and tumor recurrence in bladder cancer. We identified a positive correlation of Oct4 expression with tumor recurrence in 122 clinical specimens of superficial high-grade (stages T1-2) bladder transitional cell carcinoma (TCC). Increased Oct4 levels in bladder tumors were associated with short recurrence-free intervals in the patients. Chemotherapy induced Oct4 expression in bladder cancer cells. Notably, treatment with cisplatin increased CD44-positive bladder cancer cells expressing Oct4, representing cancer stem-like cell subpopulation. Forced expression of Oct4 reduced, whereas knockdown of Oct4 enhanced, drug sensitivity in bladder cancer cells. Furthermore, tumor cells overexpressing Oct4 responded poorly to cisplatin in vivo. In regard to clinical relevance, inhibition of Oct4 by all-trans retinoic acid (ATRA) synergistically increased sensitivity to cisplatin in bladder cancer cells. Furthermore, the combination of cisplatin and ATRA was superior to cisplatin alone in suppressing tumor growth. Therefore, our results provide evidence that Oct4 increases drug resistance and implicate that inhibition of Oct4 may be a therapeutic strategy to circumvent drug resistance.

Etoposide enhances antitumor efficacy of MDR1-driven oncolytic adenovirus through autoupregulation of the MDR1 promoter activity.

Conditionally replicating adenoviruses (CRAds), or oncolytic adenoviruses, such as E1B55K-deleted adenovirus, are attractive anticancer agents. However, the therapeutic efficacy of E1B55K-deleted adenovirus for refractory solid tumors has been limited. Environmental stress conditions may induce nuclear accumulation of YB-1, which occurs in multidrug-resistant and adenovirus-infected cancer cells. Overexpression and nuclear localization of YB-1 are associated with poor prognosis and tumor recurrence in various cancers. Nuclear YB-1 transactivates the multidrug resistance 1 (MDR1) genes through the Y-box. Here, we developed a novel E1B55K-deleted adenovirus driven by the MDR1 promoter, designed Ad5GS3. We tested the feasibility of using YB-1 to transcriptionally regulate Ad5GS3 replication in cancer cells and thereby to enhance antitumor efficacy. We evaluated synergistic antitumor effects of oncolytic virotherapy in combination with chemotherapy. Our results show that adenovirus E1A induced E2F-1 activity to augment YB-1 expression, which shut down host protein synthesis in cancer cells during adenovirus replication. In cancer cells infected with Ad5WS1, an E1B55K-deleted adenovirus driven by the E1 promoter, E1A enhanced YB-1 expression, and then further phosphorylated Akt, which, in turn, triggered nuclear translocation of YB-1. Ad5GS3 in combination with chemotherapeutic agents facilitated nuclear localization of YB-1 and, in turn, upregulated the MDR1 promoter activity and enhanced Ad5GS3 replication in cancer cells. Thus, E1A, YB-1, and the MDR1 promoter form a positive feedback loop to promote Ad5GS3 replication in cancer cells, and this regulation can be further augmented when chemotherapeutic agents are added. In the in vivo study, Ad5GS3 in combination with etoposide synergistically suppressed tumor growth and prolonged survival in NOD/SCID mice bearing human lung tumor xenografts. More importantly, Ad5GS3 exerted potent oncolytic activity against clinical advanced lung adenocarcinoma, which was associated with elevated levels of nuclear YB-1 and cytoplasmic MDR1 expression in the advanced tumors. Therefore, Ad5GS3 may have therapeutic potential for cancer treatment, especially in combination with chemotherapy. Because YB-1 is expressed in a broad spectrum of cancers, this oncolytic adenovirus may be broadly applicable.