Anti-tumor effects of deubiquitinating enzyme inhibitor PR-619 in human chondrosarcoma through reduced cell proliferation and endoplasmic reticulum stress-related apoptosis.

Chondrosarcoma, a treatment-resistant cancer with limited therapeutic options, lacks significant advancements in treatment methods. However, PR-619, a novel inhibitor of deubiquitinating enzymes, has demonstrated anti-tumor effects in various malignancies. This study aimed to investigate the impact of PR-619 on chondrosarcoma both in vitro and in vivo. Two human chondrosarcoma cell lines, SW11353 and JJ012, were utilized. Cell viability was assessed using an MTT assay, while flow cytometry enabled the detection of apoptosis and cell cycle progression. Western blotting analyses were conducted to evaluate apoptosis, cell stress, and endoplasmic reticulum (ER) stress. Furthermore, the in vivo anti-tumor effects of PR-619 were examined using a xenograft mouse model. The results revealed that PR-619 induced cytotoxicity, apoptosis, and cell cycle arrest at the G0/G1 stage by activating caspases, PARP cleavage, and p21. Moreover, PR-619 increased the accumulation of polyubiquitinated proteins and ER stress by activating IRE1, GRP78, caspase-4, CHOP, and other cellular stress responses, including JNK activation. In vivo analysis demonstrated that PR-619 effectively inhibited tumor growth with minimal toxicity in the xenograft mouse model. These findings provide evidence of the anti-tumor effects and induction of cellular and ER stress by PR-619 in human chondrosarcoma, suggesting its potential as a novel therapeutic strategy for in human chondrosarcoma.

PR-619, a General Inhibitor of Deubiquitylating Enzymes, Diminishes Cisplatin Resistance in Urothelial Carcinoma Cells through the Suppression of c-Myc: An In Vitro and In Vivo Study.

Cisplatin-based chemotherapy is the standard treatment for bladder urothelial carcinoma (UC). Most patients experience chemoresistance, the primary cause of treatment failure, which leads to disease relapse. The underlying mechanism of chemoresistance involves reduced apoptosis. In this study, we investigated the antitumor effect of the deubiquitylating enzyme inhibitor PR-619 in cisplatin-resistant bladder UC. Deubiquitinase (ubiquitin-specific protease 14 (USP14) and USP21) immunohistochemical staining demonstrated that deubiquitination is related to chemoresistance in patients with metastatic UC and may be a target for overcoming chemoresistance. Cytotoxicity and apoptosis were assessed using fluorescence-activated flow cytometry and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assay, and PR-619 was found to enhance the cytotoxic and apoptotic effects of cisplatin in cisplatin-resistant T24/R cells. Mitigated cisplatin chemoresistance was associated with the concurrent suppression of c-Myc expression in T24/R cells. Moreover, the expression of c-Myc was upregulated in human bladder UC specimens from patients with chemoresistance. Experiments in a xenograft nude mouse model confirmed that PR-619 enhanced the antitumor effects of cisplatin. These results are promising for the development of therapeutic strategies to prevent UC chemoresistance through the combined use of chemotherapeutic agents/deubiquitination inhibitors (PR-619) by targeting the c-Myc pathway.

THZ1, a covalent CDK7 inhibitor, enhances gemcitabine-induced cytotoxicity via suppression of Bcl-2 in urothelial carcinoma.

Chemotherapy with gemcitabine plus cisplatin remains the mainstay of treatment for metastatic urothelial carcinoma (UC); however, drug resistance occurs in most patients and eventually leads to treatment failure. In this study, we investigated the role of cyclin-dependent kinase 7 (CDK7) regulation in the treatment of human UCs. Moreover, we studied the effect of THZ1, a CDK7 inhibitor, alone and in combination with gemcitabine, on UCs and explored the underlying mechanism. Immunohistochemical staining showed that CDK7 expression was significantly higher in UC tumors than in counterpart urothelium. THZ1 elicited dose-dependent cytotoxicity and apoptosis in two high-grade UC cells (BFTC905 and T24). THZ1 co-treatment potentiated gemcitabine-induced cytotoxicity with suppression of B-cell lymphoma 2 (Bcl-2). Studies with a xenograft nude mouse model also confirmed that THZ1 enhanced the antitumor effect of gemcitabine on UC. These findings provide important pilot data to target CDK7 or Bcl-2 for the treatment of UCs and for overcoming chemoresistance in UCs.

Neddylation inhibitor, MLN4924 suppresses angiogenesis in huvecs and solid cancers: in vitro and in vivo study.

Cullin-RING E3 ligases are involved in the ubiquitination of substrates that regulate important biological processes and are a potential therapeutic target in many types of cancer. MLN4924, a small molecule of NEDD8-activating enzyme inhibitor, inactivates CRL by blocking cullin neddylation and has been reported to elicit anti-tumor effect. In this study, In this study, we aimed to investigate the effects of MLN4924 on angiogenesis in human umbilical vascular endothelial cells (HUVECs) and four types of cancer cells. Our results showed that MLN4924 inhibits cell viability and induced apoptosis in HUVECs in a dose-dependent manner. MLN4924 inhibits proliferation and interferes with the cell cycle checkpoint regulators, p21, p27, and phospho-histone H3. Vascular endothelial growth factor (VEGF) treatment increased the level of UBC12 in HUVECs, indicating that neddylation pathway is involved in VEGF-activated angiogenesis. MLN4924 decreased VEGF-activated cell proliferation via neddylation inhibition. MLN4924 inhibited VEGF-activated cell migration, capillary tube formation and VEGF-mediated Erk1/2 activation in HUVECs. We also examined antitumor effect of MLN4924 using xenograft SCID mouse models of four different types of cancer cells. The in vivo results showed MLN4924 inhibited tumor growth in all four types of cancers with decreasing CD31 expression in xenograft tumor. In conclusion, MLN4924 inhibited viability, migration, and VEGF-promoted angiogenic activity in HUVECs; consistently, MLN4924 inhibited tumor growth in four types of cancers with suppression of angiogenesis. These findings provide evidence to develop therapeutic strategy for cancer treatment through anti-angiogenesis through neddylation inhibition.

Suppression of Angiogenesis by Targeting Cyclin-Dependent Kinase 7 in Human Umbilical Vein Endothelial Cells and Renal Cell Carcinoma: An In Vitro and In Vivo Study.

Cancer cells rely on aberrant transcription for growth and survival. Cyclin-dependent kinases (CDKs) play critical roles in regulating gene transcription by modulating the activity of RNA polymerase II (RNAPII). THZ1, a selective covalent inhibitor of CDK7, has antitumor effects in several human cancers. In this study, we investigated the role and therapeutic potential of CDK7 in regulating the angiogenic activity of endothelial cells and human renal cell carcinoma (RCC). Our results revealed that vascular endothelial growth factor (VEGF), a critical activator of angiogenesis, upregulated the expression of CDK7 and RNAPII, and the phosphorylation of RNAPII at serine 5 and 7 in human umbilical vein endothelial cells (HUVECs), indicating the transcriptional activity of CDK7 may be involved in VEGF-activated angiogenic activity of endothelium. Furthermore, through suppressing CDK7 activity, THZ1 suppressed VEGF-activated proliferation and migration, as well as enhanced apoptosis of HUVECs. Moreover, THZ1 inhibited VEGF-activated capillary tube formation and CDK7 knockdown consistently diminished tube formation in HUVECs. Additionally, THZ1 reduced VEGF expression in human RCC cells (786-O and Caki-2), and THZ1 treatment inhibited tumor growth, vascularity, and angiogenic marker (CD31) expression in RCC xenografts. Our results demonstrated that CDK7-mediated transcription was involved in the angiogenic activity of endothelium and human RCC. THZ1 suppressed VEGF-mediated VEGFR2 downstream activation of angiogenesis, providing a new perspective for antitumor therapy in RCC patients.

The Deubiquitinating Enzyme Inhibitor PR-619 Enhances the Cytotoxicity of Cisplatin via the Suppression of Anti-Apoptotic Bcl-2 Protein: In Vitro and In Vivo Study.

After chemotherapy for the treatment of metastatic bladder urothelial carcinoma (UC), most patients inevitably encounter drug resistance and resultant treatment failure. Deubiquitinating enzymes (DUBs) remove ubiquitin from target proteins and play a critical role in maintaining protein homeostasis. This study investigated the antitumor effect of PR-619, a DUBs inhibitor, in combination with cisplatin, for bladder UC treatment. Our results showed that PR-619 effectively induced dose- and time-dependent cytotoxicity, apoptosis, and ER-stress related apoptosis in human UC (T24 and BFTC-905) cells. Additionally, co-treatment of PR-619 with cisplatin potentiated cisplatin-induced cytotoxicity in UC cells and was accompanied by the concurrent suppression of Bcl-2. We also proved that Bcl-2 overexpression is related to the chemo-resistant status in patients with metastatic UC by immunohistochemistry (IHC) staining. In a xenograft mice model, we confirmed that PR-619 enhanced the antitumor effect of cisplatin on cisplatin-naïve and cisplatin-resistant UCs. Our results demonstrated that PR-619 effectively enhanced the cisplatin-induced antitumor effect via concurrent suppression of the Bcl-2 level. These findings provide promising insight for developing a therapeutic strategy for UC treatment.

Trifluoperazine, an Antipsychotic Drug, Effectively Reduces Drug Resistance in Cisplatin-Resistant Urothelial Carcinoma Cells via Suppressing Bcl-xL: An In Vitro and In Vivo Study.

Cisplatin-based chemotherapy is the primary treatment for metastatic bladder urothelial carcinoma (UC). Most patients inevitably encounter drug resistance and resultant disease relapse. Reduced apoptosis plays a critical role in chemoresistance. Trifluoperazine (TFP), an antipsychotic agent, has demonstrated antitumor effects on various cancers. This study investigated the efficacy of TFP in inhibiting cisplatin-resistant bladder UC and explored the underlying mechanism. Our results revealed that cisplatin-resistant UC cells (T24/R) upregulated the antiapoptotic factor, B-cell lymphoma-extra large (Bcl-xL). Knockdown of Bcl-xL by siRNA resensitized cisplatin-resistant cells to the cisplatin cytotoxic effect. TFP (10-45 µM) alone elicited dose-dependent cytotoxicity, apoptosis, and G0/G1 arrest on T24/R cells. Co-treatment of TFP potentiated cisplatin-induced cytotoxicity in T24/R cells. The phenomenon that TFP alleviated cisplatin resistance to T24/R was accompanied with concurrent suppression of Bcl-xL. In vivo models confirmed that TFP alone effectively suppressed the T24/R xenograft in nude mice. TFP co-treatment enhanced the antitumor effect of cisplatin on the T24/R xenograft. Our results demonstrated that TFP effectively inhibited cisplatin-resistant UCs and circumvented cisplatin resistance with concurrent Bcl-xL downregulation. These findings provide a promising insight to develop a therapeutic strategy for chemoresistant UCs.

Histone Deacetylase Inhibitor, Trichostatin A, Synergistically Enhances Paclitaxel-Induced Cytotoxicity in Urothelial Carcinoma Cells by Suppressing the ERK Pathway.

Trichostatin A (TSA), an antifungal antibiotic derived from Streptomyces, inhibits mammalian histone deacetylases, and especially, selectively inhibits class I and II histone deacetylase (HDAC) families of enzymes. TSA reportedly elicits an antiproliferative response in multifarious tumors. This study investigated the antitumor effects of TSA alone and in combination with paclitaxel when applied to two high-grade urothelial carcinoma (UC) cell lines (BFTC-905 and BFTC-909). Fluorescence-activated cell sorting, flow cytometry, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assay were used to assess TSA's cytotoxicity and effects on apoptosis induction. TSA induced synergistic cytotoxicity, when combined with paclitaxel (combination index < 1), resulted in concomitant suppression of paclitaxel-induced activation of phospho-extracellular signal-regulated kinase (ERK) 1/2. A xenograft nude mouse model confirmed that TSA enhances the antitumor effects of paclitaxel. These findings demonstrate that the administration of TSA in combination with paclitaxel elicits a synergistic cytotoxic response. The results of this study indicate that the chemoresistance of UC could be circumvented by combining HDAC inhibitors to target the ERK pathway.

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

In Fig. 1b, upper part, the cell viability counts after treatment with cisplatin and TSA in T24 cells was by mistake a duplication of the image for NTUB1 on the left. In the corrected version of Fig. 1, the image was replaced appropriately.

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.

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.

MLN4924, a Novel NEDD8-activating enzyme inhibitor, exhibits antitumor activity and enhances cisplatin-induced cytotoxicity in human cervical carcinoma: in vitro and in vivo study.

MLN4924, an inhibitor of NEDD8 activating enzyme (NAE), has been reported to have activity against various malignancies. Here, we investigated the antitumor properties of MLN4924 and MLN4924 in combination with cisplatin on human cervical carcinoma (CC) in vitro and in vivo. Two human CC cell lines, ME-180 and HeLa, were used in this study. The cytotoxic effects of MLN4924 and/or cisplatin were measured by cell viability (MTT), proliferation (BrdU incorporation), apoptosis (flow cytometry with annexin V-FITC labeling), and the expression of cell apoptosis-related proteins (Western blotting). In vivo efficacy was determined in Nu/Nu nude mice with ME-180 and HeLa xenografts. The results showed that MLN4924 elicited viability inhibition, anti-proliferation and apoptosis in human CC cells, accompanied by activations of apoptosis-related molecules and Bid, Bcl-2 phosphorylation interruption, and interference with cell cycle regulators. Moreover, MLN4924 caused an endoplasmic reticulum stress response (caspase-4, ATF-4 and CHOP activations) and expression of other cellular stress molecules (JNK and c-Jun activations). Additionally, MLN4924 suppressed growth of CC xenografts in nude mice. Furthermore, we demonstrated that MLN4924 potentiated cisplatin-induced cytotoxicity in CC cells with activation of caspases. Consistently with this, MLN4924 significantly enhanced cisplatin-induced growth inhibition of CC xenografts. Together, these findings suggest that MLN4924 alone or in combination with cisplatin is of value in treating human CCs.