New frontiers against sorafenib resistance in renal cell carcinoma: From molecular mechanisms to predictive biomarkers.

Renal cell carcinoma (RCC) is a highly vascularized tumor and prone to distant metastasis. Sorafenib is the first targeted multikinase inhibitor and first-line chemical drug approved for RCC therapy. In fact, only a small number of RCC patients benefit significantly from sorafenib treatment, while the growing prevalence of sorafenib resistance has become a major obstacle for drug therapy effectivity of sorafenib. The molecular mechanisms of sorafenib resistance in RCC are not completely understood by now. Herein, we comprehensively summarize the underlying mechanisms of sorafenib resistance and molecular biomarkers for predicting sorafenib responsiveness. Moreover, we outline strategies suitable for overcoming sorafenib resistance and prospect potential approaches for identifying biomarkers associated with sorafenib resistance in RCC, which contributes to guide individualized and precision drug therapy.

Inhibitory effect of Ubenimex combined with fluorouracil on multiple drug resistance and P-glycoprotein expression level in non-small lung cancer.

Tumour drug resistance is one of the most urgent issues faced by anti-tumour therapies. P-glycoprotein (P-gp) has been reported to be correlated with drug resistance. In this study, we aimed to study the synergistic effect of fluorouracil (5FU) and Ubenimex (UBE) on drug resistance in lung cancer. In this study, the tumour inhibitory role of 5FU and UBE was assessed in nude mice bearing A549 or A549/ADR. Real-time polymerase chain reaction, Western blot and immunohistochemical were performed to analyse the mRNA and protein expression of P-gp. TUNEL assay was used to evaluate the apoptosis of A549/ADR cells under 5FU and UBE treatment. MTT assay was performed to calculate the IC50 value of 5FU and UBE in A549 or A549/ADR. Combined administration of 5FU and UBE significantly inhibited the tumour growth of multidrug-resistant cell lines A549/ADR in nude mice by down-regulating the mRNA and protein expression of P-gp. The apoptosis of A549/ADR was remarkably elevated in nude mice treated with 5FU and UBE. The IC50 value of 5FU and UBE was dramatically declined in A549/ADR cells compared with that of 5FU or UBE alone. Combined treatment of 5FU and UBE remarkably enhanced the apoptosis of A549/ADR cells by enhancing the intracellular accumulation of the drugs. The results of this study demonstrated that UBE combined with fluorouracil attenuated multiple drug resistance and inhibited the expression of P-gp in lung cancer.

MicroRNA-125a attenuates the chemoresistance against ubenimex in non-small cell lung carcinoma via targeting the aminopeptidase N signaling pathway.

BACKGROUND: Since several long noncoding RNAs (lncRNAs) have been implicated in the development of chemoresistance in non-small cell lung carcinoma (NSCLC), the aim of this study was to investigate whether antisense noncoding RNA in the INK4 locus (ANRIL) was associated with the chemoresistance of NSCLC. METHOD: Real-time polymerase chain reaction was performed to identify potential lncRNAs involved in the chemoresistance of NSCLC, while in-silicon analyses and luciferase assays were carried out to explore the regulatory relationship among ANRIL, miR-125a, and aminopeptidase N (APN). RESULTS: Ubenimex resistant cells were associated with a high expression of ANRIL, which directly binds to miR-125a. MiR-125a directly targeted APN expression. In addition, miR-125a and ANRIL small interfering RNA inhibited the expression of APN but promoted the expression of beclin-1 and LC3, whereas ANRIL, by competing with miR-125a, promoted cell proliferation and inhibited cell apoptosis. CONCLUSION: The data of this study suggested that, by targeting ANRIL and the APN signaling pathway, miR-125a inhibited the proliferation of NSCLC cells and promoted their apoptosis, thus attenuating the chemoresistance of NSCLC against Ubenimex.

New Method for Detecting the Suppressing Effect of Enzyme Activity by Aminopeptidase N Inhibitor.

Aminopeptidase N, also known as CD13, is a transmembrance protease with many functions. CD13 is involved in inflammatory diseases and cancers. A convenient and reliable laboratory test method for detecting the suppressing effects of enzyme activity would be useful for study of CD13 inhibitors. Porcine CD13 (pCD13) was traditionally considered an enzyme source but has significant practical disadvantages. pCD13 is not a human source, and the accuracy and reliability of experimental results are greatly reduced. In this study, a modified detection method with K562-CD13 monoclonal cells, a human-derived cell line, was established to detect the suppressing effects of enzyme activity by the CD13 inhibitor. In this method, K562-CD13 monoclonal cells were used as enzyme source and L-leucine p-nitroaniline hydrochloride as substrate. Using CD13 enzyme activity analyses, we found that the ability of the catalytic substrate was weaker in K562 cells than in the other cell lines, and K562-CD13 cells expressed significantly higher levels of CD13 enzyme activity than parental K562 cells. The enzyme activity of CD13 was detected with the new method after ubenimex treatment. The enzyme activity was significantly inhibited by ubenimex in a dose-dependent manner. In summary, this study proposes a sensitive, stable, and objective laboratory method for detecting the inhibitory effect of the CD13 inhibitor.

Discovery of a novel chimeric ubenimex-gemcitabine with potent oral antitumor activity.

Herein, a novel mutual prodrug BC-A1 was discovered by integrating ubenimex and gemcitabine into one molecule. Biological characterization revealed that compound BC-A1 could maintain both the anti-CD13 activity of ubenimex and the cytotoxic activity of gemcitabine in vitro. Further characterization also demonstrated that compound BC-A1 exhibited significant anti-invasion and anti-angiogenesis effects in vitro. The preliminary stability test of BC-A1 revealed that it could release gemcitabine in vitro. The in vivo anti-tumor results in liver cancer showed that at the same dosage, oral administration of BC-A1 was as potent as intraperitoneal administration of gemcitabine. This warranted the further research and development of the orally active prodrug BC-A1 because gemcitabine can not be orally administrated in clinic.

Ubenimex combined with Albendazole for the treatment of Echinococcus multilocularis-induced alveolar echinococcosis in mice.

Introduction: Alveolar echinococcosis (AE) is a parasitic disease caused by E. multilocularis metacestodes and it is highly prevalent in the northern hemisphere. We have previously found that vaccination with E. multilocularis-Leucine aminopeptidase (EM-LAP) could inhibit the growth and invasion of E. multilocularis in host liver, and Ubenimex, a broad-spectrum inhibitor of LAP, could also inhibit E. multilocularis invasion but had a limited effect on the growth and development of E. multilocularis. Methods: In this study, the therapeutic effect of Ubenimex combined with Albendazole on AE was evaluated. Mice were intraperitoneally injected with protoscoleces and imaging examination was performed at week 8 and week 16 to detect cyst change. During this period, mice were intraperitoneally injected with Ubenimex and intragastrically administered with Albendazole suspension. At last, the therapeutic effect was evaluated by morphological and pathological examination and liver function. Results: The results revealed that the combined treatment could inhibit the growth and infiltration of cysts in BALB/c mice infected with E. multilocularis protoscoleces. The weight, number, invasion and fibrosis of cysts were reduced in mice treated with Ubenimex in combination with Albendazole. The same effect was achieved by the single Ubenimex treatment because of its inhibitory effect on LAP activity, but it was less effective in inhibiting the growth of cysts. The levels of ALT, AST, TBIL, DBIL, ALP, and γ-GT were reduced after the combined treatment, indicating that treatment with both Ubenimex and Albendazole could alleviate liver damage. Discussion: This study suggests that the combined treatment with Ubenimex and Albendazole could be a potential therapeutic strategy for E. multilocularis infections.

A Phase â ¡ Study of Ubenimex Combined With Pembrolizumab, Nab-Paclitaxel, and Carboplatin for Previously Untreated Advanced Squamous Non-Small-Cell Lung Cancer: TORG2241 (UBE-Q).

BACKGROUND: According to the results of the KEYNOTE-407 trial, pembrolizumab plus platinum-based chemotherapy is the standard of care for patients with previously untreated advanced squamous non-small-cell lung cancer (NSCLC). Ubenimex, a potent aminopeptidase inhibitor, is an oral drug with immunostimulatory and antitumor activities. We aim to assess the safety and efficacy of ubenimex in combination with pembrolizumab, nab-paclitaxel, and carboplatin in patients with previously untreated advanced squamous NSCLC. PATIENTS AND METHODS: This prospective, single-arm, multicenter, phase II clinical trial is conducted to confirm the tolerability and efficacy of the tested drugs. Patients with previously untreated advanced squamous NSCLC will receive a predetermined daily dose of ubenimex orally plus 4 cycles of pembrolizumab, nab-paclitaxel, and carboplatin, followed by continuous administration of ubenimex and pembrolizumab for a maximum of 2 years. To confirm tolerability, the daily dose of ubenimex will begin at level 1 (30 mg), which will be increased to levels 2 (60 mg) and 3 (120 mg) according to the escalation criteria, with a standard 3 + 3 design for achieving the target dose-limiting toxicity rate of 33%. The efficacy, safety, and tolerability of ubenimex at the determined dose level will be analyzed. The primary endpoint of the efficacy evaluation will be the objective response rate assessed by an independent review committee. CONCLUSIONS: This is the first study to evaluate the efficacy and safety of ubenimex combined with pembrolizumab, nab-paclitaxel, and carboplatin in patients with previously untreated advanced squamous NSCLC. The results will help devise future treatment strategies.

Ubenimex suppresses glycolysis mediated by CD13/Hedgehog signaling to enhance the effect of cisplatin in liver cancer.

Background: Liver cancer ranks third in fatalities among all cancer-related deaths. As a traditional chemotherapy drug, the application of cis-Diamminedichloroplatinum (II) (cisplatin, CDDP) for the treatment of liver cancer is greatly limited by its side effects and high drug resistance. Therefore, we are in urgent need of a more effective and less toxic CDDP therapeutic regimen. Our research aimed to clarify the possible mechanism of ubenimex in enhancing the effect of CDDP on liver cancer. Methods: The underlying mechanism was determined using Cell Counting Kit-8 (CCK-8) assay, flow cytometry, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), transwell assay, wound healing assay and western blot assay. Results: The data indicated that ubenimex suppressed the expression levels of glycolysis-related proteins by decreasing the expression levels of cluster of differentiation 13 (CD13), while overexpression of CD13 could restore the activity of glycolysis. The glycolysis inhibitor 2-deoxy-D-glucose enhanced the antiproliferative effect of ubenimex and CDDP. In addition, the inhibition of the activity levels of the Hedgehog (Hh) pathway members was accompanied by a decrease in CD13 expression, which was reversed following CD13 overexpression. Moreover, ubenimex inhibited the production of lactic acid and adenosine triphosphate (ATP), as well as the expression of key proteins involved in glycolysis, which was similar to the effects caused by the Hh inhibitor cyclopamine. However, the effects of ubenimex were mediated by targeting CD13, while cyclopamine exhibited no effects on CD13, suggesting that Hh signaling occurred in the downstream of CD13. The inhibition of glycolysis by cyclopamine was reduced following CD13 overexpression, which further indicated that ubenimex targeted the CD13/Hh pathway to inhibit glycolysis. Finally, wound healing and transwell assays and cell proliferation and apoptosis analysis demonstrated that ubenimex inhibited glycolysis by alleviating the CD13/Hh pathway, which in turn enhanced the effects of CDDP on inhibiting the progression of liver cancer. Conclusions: Ubenimex inhibits glycolysis by targeting the CD13/Hh pathway, thus playing an anti-tumor role together with CDDP. This study demonstrated the adjuvant effect of ubenimex from the perspective of Hh signal-dependent glycolysis regulation.

Therapeutic effect on Alveolar echinococcosis by targeting EM-Leucine aminopeptidase.

Alveolar echinococcosis (AE) is a parasitic disease caused by E. multilocularis metacestodes and it is highly prevalent in the northern hemisphere. We have previously found that vaccination with E. multilocularis Leucine aminopeptidase (EM-LAP) induced specific immune response and had an inhibiting effect on the parasites. In this study, the therapeutic effect of recombinant EM-LAP (rEM-LAP) on AE was evaluated and verified using Ubenimex, a broad-spectrum inhibitor of LAP. The results reveal that rEM-LAP could inhibit cyst growth and invasion and induce specific immunity response in BALB/c mice infected with E. multilocularis protoscoleces. The ultrasonic, MRI, and morphological results show that treatment with rEM-LAP inhibits E. multilocularis infection and reduces cyst weight, number, fibrosis and invasion. The same effect is observed for the treatment with Ubenimex by inhibiting LAP activity. The indirect ELISA shows that rEM-LAP could induce specific immunity response and produce high levels of IgG, IgG1, IgG2a, IgM, and IgA, and the serum levels of IFN-γ and IL-4 are significantly increased compared to the control groups, indicating that treatment with rEM-LAP leads to a Th1 and Th2 mixed-type immune response. This study suggests that EM-LAP could be a potential therapeutic target of E. multilocularis infection.

CD13 downregulation mediated by ubenimex inhibits autophagy to overcome 5-FU resistance by disturbing the EMP3/FAK/NF-κB pathway in gastric cancer cells.

Background: Gastric cancer (GC) is one of the most common malignant tumours in China, but the efficacy of chemotherapy on GC is significantly reduced due to the occurrence of drug resistance. Some studies have shown that the expression level of CD13 is associated with tumour resistance, but whether ubenimex, as a CD13 inhibitor, reverses GC drug resistance and the underlying mechanism remain unclear. Methods: Herein, resistance to 5-fluorouracil (5-FU) was reversed in GC by ubenimex, and the underlying mechanism was determined using Cell Counting Kit-8 (CCK-8) assays, gene chip analysis, high content screening (HCS), transmission electron microscopy, flow cytometry, immunofluorescence and western blot assays. Results: Flow cytometry, transmission electron microscopy and immunofluorescence analyses indicated that ubenimex, an inhibitor of CD13, regulated the autophagy and apoptosis of SGC7901/5-FU cells by downregulating CD13 expression. In addition, Gene chip analysis and HCS demonstrated that epithelial membrane protein 3 (EMP3)/focal adhesion kinase (FAK) was a putative signalling pathway downstream of CD13. Furthermore, western blot analyses showed that ubenimex not only inhibited EMP3, FAK and nuclear factor-κB (NF-κB) expression but also suppressed GC autophagy and activated apoptosis by targeting CD13. These findings indicated a potential mechanism via the CD13/EMP3/FAK/NF-κB pathway and that the activity of which was restrained. Conclusions: Ubenimex affects autophagy and apoptosis to reverse GC cell resistance by targeting the CD13/EMP3/FAK/NF-κB pathway. These results showed that ubenimex is a promising agent that may inhibit GC autophagy to improve chemotherapeutic drug sensitivity and thereby reverse drug resistance.

Suppression of CD13 Enhances the Cytotoxic Effect of Chemotherapeutic Drugs in Hepatocellular Carcinoma Cells.

Multidrug resistance (MDR) of hepatocellular carcinoma (HCC) is a serious problem that directly hinders the effect of chemotherapeutics. In this study, we mainly explore the molecular mechanism of ROS-induced CD13 expression using hepatocarcinoma cells as the research object. We show that the drug of fluorouracil (5FU), epirubicin (EPI) and gemcitabine (GEM) can induce ROS generation, activate Ets2 and promote CD13 expression. Meanwhile, CD13 can activate NRF1 and up-regulate ROS scavenging genes transcription, such as SOD1, GPX1, GPX2 and GPX3, leading to down-regulation of intracellular ROS level and reducing the sensitivity of cells to chemotherapy agent. We also detected the anti-tumor effect of the combination therapy, CD13 inhibitor ubenimex and a variety of conventional anti-cancer drugs, such as 5FU, EPI, GEM, pemetrexed (Pem) and paclitaxel (PTX) were employed in combination. Ubenimex enhances the sensitivity of different chemotherapeutic agents and cooperates with chemotherapeutic agents to suppress tumor growth in vitro and in vivo. In general, overexpression of CD13 can lead to chemotherapy resistance, and CD13 inhibitor can reverse this effect. Combination of chemotherapy agent and ubenimex will become a potential treatment strategy for liver cancer resistance.

Ubenimex Suppresses the Ability of Migration and Invasion in Gastric Cancer Cells by Alleviating the Activity of the CD13/NAB1/MAPK Pathway.

BACKGROUND: Gastric cancer (GC) is one of the most common malignant tumors in China. Most GC patients are diagnosed at an advanced stage, for that the prognosis is dismal and metastasis is common. Although there have been increasing numbers of studies indicating that Ubenimex can suppress metastasis in GC, the underlying mechanism is still unknown. METHODS: Herein, the inhibitory effect of Ubenimex on GC metastasis, in which the underlining mechanism was determined using Gene chip analysis, high content screening (HCS), transwell assays, wound healing assays and Western blot assays. RESULTS: The results obtained from wound healing assays and transwell assays indicated that Ubenimex, an inhibitor of CD13, suppressed the migration and invasion of MKN-28, MGC-803, BGC-823 and SGC-790 cells, by downregulating CD13 expression. In addition, the findings acquired from Gene chip analysis and HCS demonstrated that NGFI-A-binding protein 1 (NAB1) was a putative target downstream of CD13. Furthermore, the results obtained from Western blot assays showed that Ubenimex not only inhibits NAB1 expression by targeting CD13, but also inhibits GC metastasis by mitigating the activity of the MAPK signaling pathway. These findings indicated a possible mechanism via the CD13/NAB1/MAPK pathway of which activity was restrained. CONCLUSION: Ubenimex exert the inhibitory effect on GC metastasis by targeting CD13, in which NAB1 expression and the activation of MAPK signaling pathway were both suppressed. This study identified a promising target for the inhibition of GC metastasis.

Ubenimex induces apoptotic and autophagic cell death in rat GH3 and MMQ cells through the ROS/ERK pathway.

PURPOSE: Ubenimex, an aminopeptidase N (APN) inhibitor, is widely known for its use as an adjunct therapy for cancer therapy. However, in recent studies, it has also conferred antitumour effects in many cancers, but its anticancer mechanism is largely unknown. This study aims to investigate the specific anticancer activities and mechanisms of ubenimex in GH3 and MMQ cells. MATERIALS AND METHODS: In this study, we investigated the anticancer effects of ubenimex in GH3 and MMQ cells. Cell viability and cell death were assessed by the Cell Counting Kit-8 kit (CCK-8) and a LIVE/DEAD cell imaging kit. Apoptosis and intracellular reactive oxygen species (ROS) generation were assessed by flow cytometry and fluorescence microscopy. Autophagosome formation was detected by transmission electron microscopy, and autophagic flux was measured with mRFP-GFP-LC3 adenoviral transfection. The protein expression level was detected by Western blotting. RESULTS: The results revealed that treatment with ubenimex induced apoptotic and autophagic cell death in GH3 and MMQ cells, which resulted in decreased viability, an increased proportion of apoptotic cells, and autophagosome formation. Further experiments showed that ubenimex induced ROS generation and activated the ROS/ERK pathway. The ROS scavenger NAC could attenuate ubenimex-induced apoptosis and autophagy. CONCLUSION: Our studies revealed that ubenimex exerted anticancer effects by inducing apoptotic and autophagic cell death in GH3 and MMQ cells, rendering it a possible effective adjunctive therapy for pituitary treatment.

Ubenimex induces autophagy inhibition and EMT suppression to overcome cisplatin resistance in GC cells by perturbing the CD13/EMP3/PI3K/AKT/NF-κB axis.

Cisplatin (CDDP)-based chemotherapy is a standard treatment for gastric cancer (GC). However, chemoresistance is a major obstacle for CDDP application. Exploring underlying mechanisms of CDDP resistance development in GC and selecting an effective strategy to overcome CDDP resistance remain a challenge. Here, we demonstrate that a transmembrane ectoenzyme, CD13, endows GC patients with insensitivity to CDDP and predicts an undesirable prognosis in GC patients with CDDP treatment. Similarly, CD13 expression is positively related with CDDP resistance in GC cells. A CD13 inhibitor, Ubenimex, reverses CDDP resistance and renders GC cells sensitivity to CDDP, for which CD13 reduction is essential, and epithelial membrane protein 3 (EMP3) is a putative target downstream of CD13. Furthermore, Ubenimex decreases EMP3 expression by boosting its CpG island hypermethylation for which CD13 down-regulation is required. In addition, EMP3 is a presumptive modifier by which CD13 exerts functions in the phosphoinositol 3-kinase/protein kinase B (PI3K/AKT) pathway. Ubenimex inhibits the activation of the CD13/EMP3/PI3K/AKT/NF-κB pathway to overcome CDDP resistance in GC cells by suppressing autophagy and epithelial-mesenchymal transition (EMT). Therefore, CD13 is a potential indicator of CDDP resistance formation, and Ubenimex may serve as a potent candidate for reversing CDDP resistance in GC.

Role of ubenimex as an anticancer drug and its synergistic effect with Akt inhibitor in human A375 and A2058 cells.

BACKGROUND: Malignant melanoma (MM) is a malignant tumor produced by changes in melanocytes in the skin or other organs. In the classification of skin tumor mortality, skin melanoma ranks the highest. Ubenimex, an Aminopeptidase N (APN) inhibitor, is now widely used for cancer as an adjunct therapy, conferring antitumor effects. Apoptosis and the induction of autophagy have both been found to be closely associated with tumor cell death. METHODS: In this study, the A375 and A2058 cell lines were treated with ubenimex. Cell viability was measured using the Cell Counting Kit 8 assay. Apoptosis and autophagic cell death were assessed using flow cytometry and acridine orange/ethidium bromide staining. Protein expression was assessed by Western blot analyses and immunofluorescence. Matrigel invasion and migration assays were used to examine the metastatic ability of melanoma cells. RESULTS: The results revealed that ubenimex inhibited the expression of APN in melanoma cells, which may be connected with the inhibition of metastasis. In addition, it increased melanoma cell death by inducing apoptosis and autophagic cell death. This effect was accompanied by increased levels of p-JNK. Moreover, treatment with ubenimex induced protective Akt activation, and combined use of an Akt inhibitor with ubenimex provided a better effect for inducing tumor cell death. CONCLUSION: As an effective anti-tumor drug in vitro, ubenimex might be an excellent adjunctive therapy for the treatment of melanoma, with greater effects when combined with the use of an Akt inhibitor.

Ubenimex, an APN inhibitor, could serve as an anti­tumor drug in RT112 and 5637 cells by operating in an Akt­associated manner.

Bladder cancer, a common urinary tract tumor, has high mortality and recurrence rates associated with metastasis. Aminopeptidase N (APN) expression and metastasis have been indicated to be associated with one another. Ubenimex may function as an APN inhibitor to inhibit the degradation of the extracellular matrix during tumorigenesis. Furthermore, APN has been widely used as an adjuvant therapy for the treatment of tumors; however, little information is available regarding the impact of ubenimex on patients. Autophagy is suggested to be important in the transformation and progression of cancer. Additionally, apoptosis, which leads to the rapid demolition of cellular organelles and structures, has also been suggested as an important factor. Thus, the present study investigated the role of ubenimex in inhibiting migration and invasion by downregulating APN expression levels to induce autophagic cell death and apoptosis in bladder cancer cells. RT112 and 5637 cell lines were treated with varying doses of ubenimex. Cell viability was measured by CCK8 colorimetry and flow cytometry. Using fluorescence microscopy, autophagic cell death was assessed using acridine orange/ethidium bromide staining. Furthermore, apoptotic cell death was assessed using flow cytometry and Trypan blue staining was used to evaluate the cell death rate. Protein expression was determined by western blot analysis. Matrigel invasion assays were exploited to assess the invasion capabilities of 5637 cells. Wound­healing migration assays and Matrigel migration assays were exploited to assess the migratory abilities of 5637 cells. Treatment with ubenimex was accompanied by decreased Akt expression, indicating that ubenimex may have similar functions to Akt inhibitors. Results also indicated that ubenimex inhibited cell migration and invasion in bladder cancer cells. Furthermore, ubenimex also induced autophagic cell death and apoptosis, which suggested that mixed programmed cell death occurred in ubenimex­treated bladder cancer cells. The results from the present study suggest that ubenimex may be a potential adjuvant therapy for the treatment of bladder cancer.

Autophagy flux inhibition, G2/M cell cycle arrest and apoptosis induction by ubenimex in glioma cell lines.

This study aimed to investigate whether ubenimex could work as an anti-tumor drug alone in glioma cells and figure out the underlying potential mechanisms. Ubenimex is widely used as an adjunct therapy in multiple solid cancers. However, it is rarely used to treat glioblastoma. The function of ubenimex in enhancing JQ1 treatment sensitivity of glioma cells by blocking autophagic degradation of HEXIM1 was previously studied. However, the detailed mechanism of autophagy regulation by ubenimex remains unclear. The U87 and U251 cell lines were treated with different doses of ubenimex. Cell viability was measured by using the WST-8 assay. Cell death was assessed using trypan blue staining and flow cytometry. The migration and invasive ability of glioma cells were examined by transwell migration/invasion assay. LC3-GFP-RFP was used to measure autophagic flux. Protein expression was assessed by Western blot analysis. Autophagosomes were evaluated using the transmission electron microscopy. Moreover, cell cycle arrest (PI Staining) was measured by flow cytometry. Results revealed that ubenimex inhibited cell proliferation as well as migration/invasion in glioma cells. Besides, ubenimex increased glioma cell death via autophagic flux inhibition. Meanwhile, ubenimex induced G2/M phase arrest and apoptosis, and this effect was accompanied by the decreased levels of p-Akt, indicating the role of ubenimex in the regulation of glioma cell proliferation and metastasis. To sum up, this study concluded that ubenimex could work as an anti-tumor drug alone in the glioma cells via inhibiting autophagic flux and inducing G2/M arrest as well as apoptosis.

Ubenimex suppresses Pim-3 kinase expression by targeting CD13 to reverse MDR in HCC cells.

Hepatocellular carcinoma (HCC) is one of the most serious cancers, with rapid progression and high mortality. However, chemotherapy of HCC is hindered by multi-drug resistance (MDR). It is urgent, therefore, to explore new approaches for overcoming MDR of HCC cells. Ubenimex, an inhibitor of CD13, has been used as an immuno-enhancer for treating hematological neoplasms and other solid tumors. Here, we demonstrate that Ubenimex can also reverse MDR in the HCC cell lines HepG2/5-FU and Bel7402/5-FU. Ubenimex inhibits the expression of the proto-oncogene, Pim-3, which is accompanied by decreased expression of BCL-2 and BCL-XL, decreased phosphorylation of Bad, and increased tumor apoptosis. Moreover, Ubenimex decreases expression of the MDR-associated proteins P-gp, MRP3 and MRP2 to enhance intracellular accumulation of Cisplatin, for which down-regulation of Pim-3 is essential. Our results reveal a previously uncharacterized function of Ubenimex in mediating drug resistance in HCC, which suggests that Ubenimex may provide a new strategy to reverse MDR and improve HCC sensitivity to chemotherapeutic drugs via its effects on Pim-3.

Ubenimex enhances the radiosensitivity of renal cell carcinoma cells by inducing autophagic cell death.

Renal cell carcinoma (RCC) is resistant to standard radiotherapy. Ubenimex, an aminopeptidase N inhibitor, is widely used as an adjunct therapy after surgery to enhance the function of immunocompetent cells and confer antitumor effects. Our previous study demonstrated that ubenimex induces autophagic cell death in RCC cells. Recently, the molecular mechanism of autophagy induction has been associated with radiosensitivity in RCC cells. In the present study, the ability of ubenimex to enhance RCC cell sensitivity to radiation via the induction of autophagic cell death was determined, and the mechanism of action of this effect was investigated. The 786-O and OS-RC-2 human RCC cell lines were treated with 0.5 mg/ml ubenimex and different doses of irradiation (IR). The cell viability was measured using a colony-formation assay and flow cytometry. Acridine orange (AO)-ethidium bromide (EB) staining was assessed by fluorescence microscopy as an indicator of autophagic cell death. Protein expression was assessed by western blotting. Autophagosomes were evaluated using transmission electron microscopy. RCC cells were used to evaluate the sensitivity to radiation using clonogenic survival and lactate dehydrogenase assays. Furthermore, these parameters were also tested at physiological oxygen levels. The AO-EB staining and flow cytometry of the OS-RC-2 cells indicated that the combined treatment significantly enhanced autophagic cell death compared with ubenimex or IR alone. Therefore, treatment with ubenimex did not significantly alter cell cycle progression but increased cell death when combined with radiation. An Akt agonist could significantly weaken this effect, indicating that ubenimex may act as an Akt inhibitor. Furthermore, the western blot analysis indicated that the combined treatment inhibited the Akt signaling pathway compared with ubenimex treatment or IR alone. Ubenimex may enhance RCC cell sensitivity to radiation by inducing cell autophagy. This induction changes the role of autophagy from protective to lethal in vitro, and this switch is associated with the inhibition of the Akt signaling pathway.

Ubenimex attenuates acquired sorafenib resistance in renal cell carcinoma by inhibiting Akt signaling in a lipophagy associated mechanism.

Sorafenib is used as first line treatment of renal cell carcinoma (RCC) due to the poor sensitivity to radiotherapy and chemotherapy of this malignancy; however, acquired resistance limits the application of sorafenib and its analogues. In this study, we explored a new strategy to overcome acquired resistance to sorafenib. The RCC cell lines 786-O and ACHN were cultured in presence of increasing concentrations of sorafenib to generate sorafenib-resistant cell lines, 786-O-R and ACHN-R. Interestingly, treatment with ubenimex (0.25 mg/ml) and 3-MA (2 mM) restored the sensitivity of resistant cell lines to sorafenib, indicating the involvement of autophagy in acquired resistance. High levels of autophagy flux were observed in resistant cells, and the opposite effects of ubenimex and 3-MA suggested a complex role for autophagy. While 3-MA abolished protection in sorafenib-resistant cells, ubenimex induced uncontrolled autophagy and autophagic cell death. Lipophagy, characterized by a lipid droplet cargo, was observed in RCC tissues and cells. In sorafenib-resistant cells, ubenimex inhibited the Akt signaling pathway that regulates autophagy. In summary, lipophagy participates in sorafenib-resistance of RCC, which could be reversed by interventions targeting the Akt pathway.