Chung-Sheng Shi

MLN4924, a novel protein neddylation inhibitor, suppresses proliferation and migration of human urothelial carcinoma: In vitro and in vivo studies

MLN4924, a small molecule inhibitor of NEDD8 activating enzyme (NAE), has been reported to elicit an anti-tumor effect on various malignancies. In this study, we investigated the anti-tumor effect of MLN4924 in human urothelial carcinoma (UC) in vitro and in vivo by using three human UC cell lines of various grading (T24, NTUB1 and RT4). The impact of MLN4924 on UC cells was determined by measuring viability (MTT), proliferation (BrdU incorporation), cell cycle progression (flow cytometry with propidium iodide staining) and apoptosis (flow cytometry with annexin V-FITC labeling). The cell cycle regulatory molecules, apoptosis-related molecules, and cell stress-related proteins were examined by Western blotting. The influence of tumor cell migration and invasion was analyzed by Transwell and wound healing assays. We also evaluated the effects of MLN4924 on tumor growth by a SCID xenograft mouse model. The data show that MLN4924 induced dose-dependent cytotoxicity, anti-proliferation, anti-migration, anti-invasion and apoptosis in human UC cells, accompanied by activations of Bad, phospho-histone H2A.X, caspase-3, 7 and PARP, decreased level of phospho-Bcl2, and caused cell cycle retardation at the G2M phase. Moreover, MLN4924 activated endoplasmic reticulum stress-related molecules (caspase-4, phospho-eIF2α, ATF-4 and CHOP) and other stress responses (JNK and c-Jun activations). Finally, we confirmed MLN4924 inhibited tumor growth in a UC xenograft mouse model with minimal general toxicity. We concluded that MLN4924 induces apoptosis and cell cycle arrest, as well as activation of cell stress responses in human UC. These findings imply MLN4924 provides a novel strategy for the treatment of UC.

2-Methoxyestradiol Induces Mitotic Arrest, Apoptosis, and Synergistic Cytotoxicity with Arsenic Trioxide in Human Urothelial Carcinoma Cells

2-Methoxyestradiol (2-ME), an endogenous derivative of 17β-estradiol, has been reported to elicit antiproliferative responses in various tumors. In this study, we investigated the effects of 2-ME on cell viability, proliferation, cell cycle, and apoptosis in human urothelial carcinoma (UC) cell lines. We used two high-grade human bladder UC cell lines (NTUB1 and T24). After treatment with 2-ME, the cell viability and apoptosis were measured by MTT assay and flow cytometry (fluorescence-activated cell sorting), with annexin V-FITC staining and propidium iodide (PI) labeling. DNA fragmentation was analyzed by agarose gel electrophoresis. Flow cytometry with PI labeling was used for the cell cycle analyses. The protein levels of caspase activations, poly (ADP-ribose) polymerase (PARP) cleavage, phospho-histone H2A.X, phospho-Bad, and cell cycle regulatory molecules were measured by Western blot. The effects of the drug combinations were analyzed using the computer software, CalcuSyn. We demonstrated that 2-ME effectively induces dose-dependent cytotoxicity and apoptosis in human UC cells after 24 h exposure. DNA fragmentation, PARP cleavage, and caspase-3, 7, 8, 9 activations can be observed with 2-ME-induced apoptosis. The decreased phospho-Bad (Ser136 and Ser155) and mitotic arrest of the cell cycle in the process of apoptosis after 2-ME treatment was remarkable. In response to mitotic arrest, the mitotic forms of cdc25C, phospho-cdc2, cyclin B1, and phospho-histone H3 (Ser10) were activated. In combination with arsenic trioxide (As2O3), 2-ME elicited synergistic cytotoxicity (combination index <1) in UC cells. We concluded that 2-ME significantly induces apoptosis through decreased phospho-Bad and arrests bladder UC cells at the mitotic phase. The synergistic antitumor effect with As2O3 provides a novel implication in clinical treatment of UC.

MLN4924 Synergistically Enhances Cisplatin-induced Cytotoxicity via JNK and Bcl-xL Pathways in Human Urothelial Carcinoma

Cisplatin-based chemotherapy is the primary treatment for metastatic bladder urothelial carcinoma. However, the response rate is only 40–65%. This study investigated the anti-tumor effect and underlying mechanisms of the combination of cisplatin and the NEDD8-activating enzyme inhibitor MLN4924 in human bladder urothelial carcinoma. The combination of cisplatin and MLN4924 exerted synergistic cytotoxicity on two high-grade bladder urothelial carcinoma cell lines, NTUB1 and T24 (combination index <1). MLN4924 also potentiated the cisplatin-induced apoptosis and activation of caspase-3 and -7, phospho-histone H2A.X and PARP. c-Jun N-terminal kinase (JNK) activation and a down-regulation of B-cell lymphoma-extra large (Bcl-xL) were also observed during cisplatin and MLN4924 treatment. Inhibition of JNK activation partially restored cell viability and Bcl-xL expression. Bcl-xL overexpression also rescued cell viability. MLN4924 significantly potentiated cisplatin-induced tumor suppression in urothelial carcinoma xenograft mice. In summary, MLN4924 synergistically enhanced the anti-tumor effect of cisplatin via an increase in DNA damage, JNK activation and down-regulation of Bcl-xL in urothelial carcinoma cells. These findings provide a new therapeutic strategy for the treatment of bladder cancer.

Epigallocatechin Gallate Attenuates Partial Bladder Outlet Obstruction-induced Bladder Injury via Suppression of Endoplasmic Reticulum Stress-related Apoptosis-In Vivo Study.

OBJECTIVES To investigate the protective effect of epigallocatechin gallate (EGCG), a green tea extract, on partial bladder outlet obstruction (pBOO)-induced bladder injury in a rat model. METHODS The female Sprague-Dawley rats underwent sham or BOO procedures, and were divided into several groups (sham with saline injection, sham with EGCG treatment, BOO with saline injection, and BOO with EGCG treatment). The rats in each group were randomized into 2 groups (48 hours and 30 days after the BOO procedure) for when their bladders were harvested. EGCG (4.5 mg/kg/day) and saline were administered via intraperitoneal injection after the BOO procedure during the study period. Bladder tissue was examined for inflammation, endoplasmic reticulum (ER) stress-related apoptotic markers by Western blot, and histological staining. RESULTS BOO induced acute bladder injury (hemorrhage, edema, and neutrophil infiltration) after 48 hours. In addition, cystometry showed a decrease in micturition pressure and intercontractile interval. We also observed increased expressions of cyclooxygenase-2, poly(ADP-ribose) polymerase at 48 hours, as well as ER stress markers such as caspase-12 and CCAAT/-enhancer-binding protein homologous protein (CHOP). Treatment with EGCG significantly improved pBOO-induced histologic changes, bladder dysfunction, and the overexpression of cyclooxygenase-2, CHOP, and caspase-12 at 48 hours. Similarly, EGCG treatment for 30 days effectively recovered compliance and intercontractile interval, submucosal ER stress-related apoptosis (CHOP and caspase-12) at 30 days after pBOO. CONCLUSIONS EGCG alleviate pBOO-induced bladder injury and dysfunction via suppression of inflammation and ER stress-related apoptosis.

The Effects of Adipose-Derived Stem Cells in a Rat Model of Tobacco-Associated Erectile Dysfunction

Tobacco use is associated with erectile dysfunction (ED) via a number of mechanisms including vascular injury and oxidative stress in corporal tissue. Adipose derived stem cells (ADSC) have been shown to ameliorate vascular/corporal injury and oxidative stress by releasing cytokines, growth factors and antioxidants. We assessed the therapeutic effects of intracavernous injection of ADSC in a rat model of tobacco-associated ED. Thirty male rats were used in this study. Ten rats exposed to room air only served as negative controls. The remaining 20 rats were passively exposed to cigarette smoke (CS) for 12 weeks. At the 12-week time point, ADSC were isolated from paragonadal fat in all rats. Amongst the 20 CS exposed rats, 10 each were assigned to one of the two following conditions: (i) injection of phosphate buffered saline (PBS) into the corpora cavernosa (CS+PBS); or (ii) injection of autologous ADSC in PBS into the corpora cavernosa (CS+ADSC). Negative control animals received PBS injection into the corpora cavernosa (normal rats [NR] + PBS). After injections all rats were returned to their previous air versus CS exposure state. Twenty-eight days after injection, all rats were placed in a metabolic cage for 24-hour urine collection to be testing for markers of oxidative stress. After 24-hour urine collection all 30 rats also underwent erectile function testing via intracavernous pressure (ICP) testing and were then sacrificed. Corporal tissues were obtained for histological assessment and Western blotting. Mean body weight was significantly lower in CS-exposed rats than in control animals. Mean ICP, ICP /mean arterial pressure ratio, serum nitric oxide level were significantly lower in the CS+PBS group compared to the NR+PBS and CS+ADSC groups. Urine markers for oxidative stress were significantly higher in the CS+PBS group compared to the NR+PBS and CS+ADSC groups. Mean expression of corporal nNOS and histological markers for endothelial and smooth muscle cells was significantly lower, and tissue apoptotic index significantly higher, in the CS+PBS group compared to the NR+PBS and CS+ADSC groups. Our findings confirm that chronic tobacco exposure causes ultrastructural damage to the corporal tissue and increases systemic oxidative stress states. Treatment with ADSC ameliorates these adverse effects and holds promise as a potential therapy for tobacco-related ED.

Piperlongumine Suppresses Proliferation of Human Oral Squamous Cell Carcinoma through Cell Cycle Arrest, Apoptosis and Senescence

Oral squamous cell carcinoma (OSCC), an aggressive cancer originating in the oral cavity, is one of the leading causes of cancer deaths in males worldwide. This study investigated the antitumor activity and mechanisms of piperlongumine (PL), a natural compound isolated from Piper longum L., in human OSCC cells. The effects of PL on cell proliferation, the cell cycle, apoptosis, senescence and reactive oxygen species (ROS) levels in human OSCC cells were investigated. PL effectively inhibited cell growth, caused cell cycle arrest and induced apoptosis and senescence in OSCC cells. Moreover, PL-mediated anti-human OSCC behavior was inhibited by an ROS scavenger N-acetyl-l-cysteine (NAC) treatment, suggesting that regulation of ROS was involved in the mechanism of the anticancer activity of PL. These findings suggest that PL suppresses tumor growth by regulating the cell cycle and inducing apoptosis and senescence and is a potential chemotherapy agent for human OSCC cells.

Trichostatin A, a histone deacetylase inhibitor, induces synergistic cytotoxicity with chemotherapy via suppression of Raf/MEK/ERK pathway in urothelial carcinoma

In this study, we aimed to investigate the antitumor effects of trichostatin A (TSA), an antifungal antibiotic that inhibits histone deacetylase (HDAC) family of enzymes, alone or in combination with anyone of the three chemotherapeutic agents (cisplatin, gemcitabine, and doxorubicin) for the treatment of human urothelial carcinoma (UC). Two high-grade human UC cell lines (T24 and NTUB1) were used. Cytotoxicity and apoptosis were assessed by MTT assay and flow cytometry, respectively. The expression of phospho-c-Raf, phospho-MEK1/2, and phospho-ERK1/2 was measured by western blotting. ERK siRNA knockdown and the specific MEK inhibitor U0126 were used to examine the role of Raf/MEK/ERK signaling pathway in combined cytotoxicity of TSA and chemotherapy. TSA co-treatment with any one of the three chemotherapeutic agents induced synergistic cytotoxicity (combination index < 1) and concomitantly suppressed chemotherapeutic drug-induced activation of Raf-MEK-ERK pathway. Combination of ERK siRNA knockdown and treatment with the specific MEK inhibitor (U0126) enhanced the cytotoxic effects of the chemotherapy on UC cells. These observations were confirmed in a xenograft nude mouse model. Moreover, activated Raf/MEK/ERK pathway was observed in human bladder UC specimens from patients with chemoresistant status. In conclusion, TSA elicits a synergistic cytotoxic response in combination with chemotherapy via targeting the Raf/MEK/ERK pathway. TSA elicits synergistic cytotoxic response in combination with three DNA-damaging drugs (cisplatin, gemcitabine, and doxorubicin). Activated Raf/MEK/ERK pathway is involved in chemoresistant mechanism of UC. Combining chemotherapeutic agents with HDAC inhibitor (TSA) or with targeting Raf/MEK/ERK pathway is promising to circumvent chemoresistance in UCs.

Abstract 5878: Trichostatin A overcomes chemotherapeutic resistance of urothelial carcinoma cells through the inactivation of c-Raf/ERK pathway

Trichostatin A (TSA), a potent histone deacetylase (HDAC) inhibitor, has been reported to elicit anti-proliferative response in various tumors. Here, we investigated antitumor effect of TSA alone or in combination with conventional chemotherapeutic agents on urothelial carcinoma (UC) cells. We use one high-grade UC cell line (T24) and another UC cells (NTUUC) obtained by primary culture from the surgical specimen of a women with high-grade and metastatic bladder UC. The cytotoxicity and apoptosis induced by TSA alone , chemotherapeutic agents (cisplatin, gemcitabine and doxorubicin) and combined treatment were assessed by MTT assay and fluorescence-activated cell sorting and flow cytometry. The expression of phosphor c-Raf, phosphor-MEK1/2, and phosphor-ERK1/2 were measured by Western blot. urther elucidation on the role of Raf/MEK/ERK pathoway on the TSA-enhanced cytoxicity were exmained by using ERK1 siRNA knockdown and the specific MEK inhibitor (PD98059). Our results showed TSA markedly enhances the cytotoxicity and apoptosis of three chemotherapeutic agents in UC cells with concurrent suppression of Raf/MEK/ERK signaling pathway. Consistently, inhibition of Down-regulation of ERK by MEK inhibitor or by ERK 1 siRNA knockdown potentiated the chemotherapeutic agnet-induced cytotoxicity of TSA in UC cells. We concluded that TSA potentiates the therapeutic efficacy of cisplatin, gemcitabine and doxorubicin in human UC cells through Raf/MEK/ERK signaling pathway. These findings provide a new treatment strategy against UC. Note: This abstract was not presented at the meeting. Citation Format: Kuan-Lin Kuo, Chung-Sheng shi, Ju-Tong Hsieh, Shiang-Peng Chen, Wei-Chou Lin, Shih-Ming Liao, Shing-Hwa Liu, Kuo-How Huang. Trichostatin A overcomes chemotherapeutic resistance of urothelial carcinoma cells through the inactivation of c-Raf/ERK pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5878. doi:10.1158/1538-7445.AM2017-5878