Peihua Luo

Oxidative stress is involved in Dasatinib-induced apoptosis in rat primary hepatocytes

Dasatinib, a multitargeted inhibitor of BCR-ABL and SRC kinases, exhibits antitumor activity and extends the survival of patients with chronic myeloid leukemia (CML) and Philadelphia chromosome-positive acute lymphoblastic leukemia (ALL). However, some patients suffer from hepatotoxicity, which occurs through an unknown mechanism. In the present study, we found that Dasatinib could induce hepatotoxicity both in vitro and in vivo. Dasatinib reduced the cell viability of rat primary hepatocytes, induced the release of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) in vitro, and triggered the ballooning degeneration of hepatocytes in Sprague-Dawley rats in vivo. Apoptotic markers (chromatin condensation, cleaved caspase-3 and cleaved PARP) were detected to indicate that the injury induced by Dasatinib in hepatocytes in vitro was mediated by apoptosis. This result was further validated in vivo using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays. Here we found that Dasatinib dramatically increased the level of reactive oxygen species (ROS) in hepatocytes, reduced the intracellular glutathione (GSH) content, attenuated the activity of superoxide dismutase (SOD), generated malondialdehyde (MDA), a product of lipid peroxidation, decreased the mitochondrial membrane potential, and activated nuclear factor erythroid 2-related factor 2 (Nrf2) and mitogen-activated protein kinases (MAPK) related to oxidative stress and survival. These results confirm that oxidative stress plays a pivotal role in Dasatinib-mediated hepatotoxicity. N-acetylcysteine (NAC), a typical antioxidant, can scavenge free radicals, attenuate oxidative stress, and protect hepatocytes against Dasatinib-induced injury. Thus, relieving oxidative stress is a viable strategy for reducing Dasatinib-induced hepatotoxicity.

Potent antitumor activity of 10-methoxy-9-nitrocamptothecin

The present data showed that 10-methoxy-9-nitrocamptothecin (MONCPT), a family of camptothecin analogues, possessed high antitumor activity in vitro and in vivo. Cytotoxicity assays showed that MONCPT was a potential and highly efficient antitumor compound with IC50 values of 0.1 to 500 nmol/L in nine tumor cell lines. The high cytotoxic potency of MONCPT was paralleled with its ability to increase the cellular accumulation of DNA damage. DNA relaxation assay also showed that MONCPT exerted high potency as a topoisomerase I inhibitor. Moreover, administration of MONCPT (5–20 mg/kg) for 15 to 17 days significantly inhibited tumor growth in human androgen–independent prostate tumor (PC3) and human non–small cell lung tumor (A549) xenografts; the inhibition rates ranged from 29.6% to 98%. The cytotoxic effect of 1,000 nmol/L of MONCPT in PC3 cells was associated with causing an arrest in G0-G1 phase, whereas that of 10 and 100 nmol/L MONCPT was relative to a persistent block in G2-M phase. Furthermore, down-regulation of CDK2, CDK4, and cyclin D1 was observed in PC3 cells treated with 1,000 nmol/L of MONCPT, whereas overexpression of CDK7, CDK1, and cyclin B1 was seen in PC3 cells treated with 10 and 100 nmol/L of MONCPT. These results suggested that cell cycle regulation might contribute to the anticancer properties of MONCPT and strongly support the further anticancer development of MONCPT. [Mol Cancer Ther 2006;5(4):962–8]

Bortezomib Sensitizes Human Acute Myeloid Leukemia Cells to All-Trans-Retinoic Acid-induced Differentiation by modifying the RARα/STAT1 axis

All-trans-retinoic acid (ATRA) has held great promise for differentiation-based therapy but reportedly downregulates retinoic acid receptor-α (RARα) in a proteasome-dependent manner, which leads to decreased acute myeloid leukemia (AML) cell differentiation efficiency. Therefore, research strategies that seek to further sensitize cells to retinoids and extend the range of retinoid-affected myeloid malignancies beyond acute promyelocytic leukemia (APL) are key investigative avenues. Here, we show that bortezomib, the first proteasome inhibitor approved for newly diagnosed and relapsed multiple myeloma, exhibited strong synergism with ATRA to promote HL60 and NB4 AML cell differentiation. We observed that bortezomib sensitized AML cells to ATRA-induced morphologic, biochemical, and functional changes, indicative of myeloid differentiation without cell death. In addition, treatment of human leukemia HL60 xenografts with bortezomib and ATRA together did not increase bortezomib-induced progressive weight loss but resulted in significant tumor growth inhibition in addition to increased differentiation (P < 0.05). These enhanced differentiation effects were accompanied by RARα stabilization and STAT1 activation. Taken together, our study was the first to evaluate bortezomib and ATRA synergy in AML cell differentiation and to assess new opportunities for bortezomib and ATRA combination as a promising approach for future differentiation therapy. Mol Cancer Ther; 12(2); 195–206. ©2012 AACR.

MEK/ERK dependent activation of STAT1 mediates dasatinib-induced differentiation of acute myeloid leukemia.

Dasatinib (BMS-354825) is a FDA-approved multitargeted kinase inhibitor of BCR/ABL and Src kinases. It is now used in the treatment of chronic myelogenous leukemia (CML) with resistance or intolerance to prior therapies, including imatinib. Here we report a novel effect of dasatinib on inducing the differentiation of acute myeloid leukemia (AML) cells through MEK/ERK-dependent activation of signal transducer and activator of transcription 1 (STAT1). We found that dasatinib could induce the differentiation of AML cells as demonstrated by the expression of differentiation marker CD11b, G0/G1 phase arrest and decreased ratio of nucleus to cytoplasm. Of note, dasatinib induced robust phosphorylation of STAT1 both at Tyr701 and Ser727 as well as the redistribution of STAT1 from the cytoplasm to the nucleus, thus leading to the transcription of STAT1-targeted genes. Knocking down STAT1 expression by shRNA significantly attenuated dasatinib-induced differentiation, indicating an important role of STAT1 in myeloid maturation. We further found that dasatinib-induced activation of STAT1 was regulated by the MEK/ERK kinases. The phosporylation of MEK and ERK occurred rapidly upon dasatinib treatment and increased progressively as differentiation was induced. MEK inhibitors PD98059 and U0216 not only inhibited the phosphorylation of STAT1, but also abrogated dasatinib-induced myeloid differentiation, suggesting that MEK/ERK dependent phosphorylation of STAT1 might be indispensable for the differentiating effect of dasatinib in AML cells. Taken together, our study suggests that STAT1 is an important mediator in dasatinib-induced differentiation of AML cells, whose activation requires the activation of MEK/ERK cascades.

Inhibition of all‐Trans‐retinoic acid‐induced proteasome activation potentiates the differentiating effect of retinoid in acute myeloid leukemia cells

All‐trans retinoic acid (ATRA) is nowadays considered to be the sole efficient agent for differentiation‐based therapy in leukemia; however, the mechanisms of ATRAs biological effects remain largely unknown. Here we first reported that ATRA‐induced myeloid leukemia differentiation was accompanied with the increased level of ubiquitin–protein conjugates and the upregulation of proteasome activity. To explore the functional role of the activated proteasome in retinoic acid (RA) signaling, the effects of proteasome inhibitors on RA‐induced cell differentiation were determined. Our results demonstrated that inhibition of ATRA‐elevated proteasome activity obviously promoted the myeloid maturation program triggered by ATRA, suggesting that the overactivated proteasome is not beneficial for ATRAs effects. Further studies demonstrated that the synergistic differentiating effects of ATRA and proteasome inhibitors might be associated with the protection of retinoic acid receptor alpha (RARα) from degradation by the ubiquitin–proteasome pathway (UPP). Moreover, the accumulated RARα was able to enhance the transcription of its target gene, which might also contribute to the enhanced differentiation of leukemia cells. Together, by linking the UPP to ATRA‐dependent signaling, our data provide a novel insight into studying the mechanisms of ATRA‐elicited cellular effects and imply the possibility of combination of ATRA and proteasome inhibitors in leukemia therapy. Mol. Carcinog.

The ubiquitin-proteasome pathway plays essential roles in ATRA-induced leukemia cells G0/G1 phase arrest and transition into granulocytic differentiation

All-trans retinoic acid (ATRA) has been successfully used in differentiation therapy for acute promyelocytic leukemia (APL) in the clinic. ATRA-induced differentiation of leukemia cells is accompanied by a G0/G1 arrest, yet how ATRA couples cell cycle arrest to differentiation remains largely unknown. Here we observed that the ubiquitin-proteasome pathway (UPP) was activated upon ATRA treatment in the human acute myeloid leukemia cell lines, NB4 and HL-60, as represented by the accumulation of ubiquitinated proteins, the up-regulation of ubiquitin mRNA and increased 20S proteasome activity. Interestingly, we found that complete inhibition of proteasome activity suppressed ATRA-induced proliferation/differentiation (P/D) transition in both cell lines. Furthermore, we demonstrate that the exact protein contributing to this phenomenon is different in these two cell lines. Cyclin-dependent kinase 2 (CDK2) and Cyclin E were degraded by the UPP; they accumulated significantly after complete inhibition of the proteasome in ATRA-treated NB4 and HL-60 cells, respectively. These findings suggested that the UPP might be indispensable in the ATRA-induced G0/G1 arrest and differentiation of leukemia cells. The exact protein degraded by the UPP to promote the myeloid maturation program set in motion by the retinoid may be cell type dependent.

Autophagy blockade sensitizes the anticancer activity of CA-4 via JNK-Bcl-2 pathway

Combretastatin A-4 (CA-4) has already entered clinical trials of solid tumors over ten years. However, the limited anticancer activity and dose-dependent toxicity restrict its clinical application. Here, we offered convincing evidence that CA-4 induced autophagy in various cancer cells, which was demonstrated by acridine orange staining of intracellular acidic vesicles, the degradation of p62, the conversion of LC3-I to LC3-II and GFP-LC3 punctate fluorescence. Interestingly, CA-4-mediated apoptotic cell death was further potentiated by pretreatment with autophagy inhibitors (3-methyladenine and bafilomycin A1) or small interfering RNAs against the autophagic genes (Atg5 and Beclin 1). The enhanced anticancer activity of CA-4 and 3-MA was further confirmed in the SGC-7901 xenograft tumor model. These findings suggested that CA-4-elicited autophagic response played a protective role that impeded the eventual cell death while autophagy inhibition was expected to improve chemotherapeutic efficacy of CA-4. Meanwhile, CA-4 treatment led to phosphorylation/activation of JNK and JNK-dependent phosphorylation of Bcl-2. Importantly, JNK inhibitor or JNK siRNA inhibited autophagy but promoted CA-4-induced apoptosis, indicating a key requirement of JNK-Bcl-2 pathway in the activation of autophagy by CA-4. We also identified that pretreatment of Bcl-2 inhibitor (ABT-737) could significantly enhance anticancer activity of CA-4 due to inhibition of autophagy. Taken together, our data suggested that the JNK-Bcl-2 pathway was considered as the critical regulator of CA-4-induced protective autophagy and a potential drug target for chemotherapeutic combination.

The Proteasome Inhibitor Bortezomib Enhances ATRA-Induced Differentiation of Neuroblastoma Cells via the JNK Mitogen-Activated Protein Kinase Pathway

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood. Differentiated human NBs are associated with better outcome and lower stage; induction of differentiation is considered to be therapeutically advantageous. All-trans retinoic acid (ATRA) has been shown to induce the differentiation of neuroblastoma (NB) cell lines. The proteasome inhibitor bortezomib inhibits cell growth and angiogenesis in NBs. Here, we investigated the synergistic effect between bortezomib and ATRA in inducing NB cell differentiation in different NB cell lines. Bortezomib combined with ATRA had a significantly enhanced antiproliferative effect. This inhibition was characterized by a synergistic increase in neuronal differentiation. At the same time, the combination therapy showed little neuronal toxicity which was assessed in primary cultures of rat cerebellar granule cells by the MTT assay, PI staining. The combination of bortezomib and ATRA triggered increased differentiation through the activation of proteins, including RARα, RARβ, RARγ, p-JNK and p21, compared with ATRA treatment alone. Using JNK inhibitor SP600125 to block JNK-dependent activity, the combination therapy-induced neuronal differentiation was partially attenuated. In addition, p21 shRNA had no effect on the combination therapy-induced neuronal differentiation. The in vivo antitumor activities were examined in human NB cell xenografts and GFP-labeled human NB cell xenografts. Treatment of human NB cell CHP126-bearing nude mice with ATRA plus bortezomib resulted in more significant tumor growth inhibition than mice treated with either drug alone. These findings provide the rationale for the development of a new therapeutic strategy for NB based on the pharmacological combination of ATRA and bortezomib.

Involvement of mitogen‐activated protein kinase in signal transducer and activator of transcription‐1 mediated differentiation induced by bortezomib in acute myeloid leukemia cells

In this paper we report a new myeloid differentiation effect of bortezomib (BTZ) in acute myeloid leukemia (AML) cell lines and primary patient‐derived AML cells; this effect was assayed by investigating growth‐inhibition, cell morphology, differentiation markers, and nitro‐blue tetrazolium reduction. We show that BTZ induces the phosphorylation of several mitogen‐activated protein (MAP) kinases, including MEK/ERK, c‐Jun N‐terminal kinase (JNK), and p38 MAPK. BTZ‐induced cell differentiation is almost completely reversed by PD98059, an inhibitor of MEK, which also attenuates the increase in phospho‐JNK p46. However, p38 activation does not appear to be required for the differentiation induced by BTZ. Furthermore, the differentiation effect of BTZ is associated with increased protein level of signal transducer and activator of transcription‐1 (STAT1), a molecular determinant of myeloid differentiation, due to effects on both its synthesis and degradation. In short, this study reveals that BTZ activates the MEK/ERK cascade, which further up‐regulates the expression and activity of the key myeloid transcription factor STAT1, thus promoting myeloid differentiation. These findings contribute to an unexpected potential mechanism for the antitumor activity of BTZ in AML.

All-trans retinoic acid synergizes with topotecan to suppress AML cells via promoting RARα-mediated DNA damage

BackgroundChemotherapy is the only therapy option for the majority of AML patients, however, there are several limitations for this treatment. Our aim was to find a new chemotherapy strategy that is more effective and less toxic.MethodsMTT assays and a xenograft mouse model were employed to evaluate the synergistic activity of all-trans retinoic acid (ATRA) combined with topotecan (TPT). Drug-induced DNA damage and apoptosis were determined by flow cytometry analysis with PI and DAPI staining, the comet assay and Western blots. Short hairpin RNA (shRNA) and a RARα plasmid were used to determine whether RARα expression influenced DNA damage and apoptosis.ResultsWe found that ATRA exhibited synergistic activity in combination with Topotecan in AML cells, and the enhanced apoptosis induced by Topotecan plus ATRA resulted from caspase pathway activation. Mechanistically, ATRA dramatically down regulated RARα protein levels and led to more DNA damage and ultimately resulted in the synergism of these two agents. In addition, the increased antitumor efficacy of Topotecan combined with ATRA was further validated in the HL60 xenograft mouse model.ConclusionsOur data demonstrated, for the first time, that the combination of TPT and ATRA showed potential benefits in AML, providing a novel insight into clinical treatment strategies.