Emodin inhibits growth and induces apoptosis in an orthotopic hepatocellular carcinoma model by blocking activation of STAT3

BACKGROUND AND PURPOSE

Aberrant activation of STAT3 is frequently encountered and promotes proliferation, survival, metastasis and angiogenesis in hepatocellular carcinoma (HCC). Here, we have investigated whether emodin mediates its effect through interference with the STAT3 activation pathway in HCC.

EXPERIMENTAL APPROACH

The effect of emodin on STAT3 activation, associated protein kinases and apoptosis was investigated using various HCC cell lines. Additionally, we also used a predictive tumour technology to analyse the effects of emodin. The in vivo effects of emodin were assessed in an orthotopic mouse model of HCC.

KEY RESULTS

Emodin suppressed STAT3 activation in a dose- and time-dependent manner in HCC cells, mediated by the modulation of activation of upstream kinases c-Src, JAK1 and JAK2. Vanadate treatment reversed emodin-induced down-regulation of STAT3, suggesting the involvement of a tyrosine phosphatase and emodin induced the expression of the tyrosine phosphatase SHP-1 that correlated with the down-regulation of constitutive STAT3 activation. Interestingly, silencing of the SHP-1 gene by siRNA abolished the ability of emodin to inhibit STAT3 activation. Finally, when administered i.p., emodin inhibited the growth of human HCC orthotopic tumours in male athymic nu/nu mice and STAT3 activation in tumour tissues.

CONCLUSIONS AND IMPLICATIONS

Emodin mediated its effects predominantly through inhibition of the STAT3 signalling cascade and thus has a particular potential for the treatment of cancers expressing constitutively activated STAT3.     

ABT-263 enhances sorafenib-induced apoptosis associated with Akt activity and the expression of Bax and p21(CIP1/WAF1) in human cancer cells

BACKGROUND AND PURPOSE

Sorafenib, a potent inhibitor that targets several kinases associated with tumourigenesis and cell survival, has been approved for clinical treatment as a single agent. However, combining sorafenib with other agents improves its anti-tumour efficacy in various preclinical tumour models. ABT-263, a second-generation BH3 mimic, binds to the anti-apoptotic family members Bcl-2, Bcl-xL and Bcl-w, and has been demonstrated to enhance TNFSF10 (TRAIL)-induced apoptosis in human hepatocarcinoma cells. Hence, we investigated the effects of ABT-263 treatment combined with sorafenib.

EXPERIMENTAL APPROACH

The effects of ABT-263 combined with sorafenib were investigated in vitro, on cell viability, clone formation and apoptosis, and the mechanism examined using western blot and flow cytometry. This combination was also evaluated in vivo, in a mouse xenograft model; tumour growth, volume and weights were measured and a TUNEL assay performed.

KEY RESULTS

ABT-263 enhanced sorafenib-induced apoptosis while sparing non-tumourigenic cells. Although ABT-263 plus sorafenib significantly stimulated intracellular reactive oxygen species production and subsequent mitochondrial depolarization, this was not sufficient to trigger cell apoptosis. ABT-263 plus sorafenib significantly decreased Akt activity, which was, at least partly, involved in its effect on apoptosis. Bax and p21 (CIP1/WAF1) were shown to play a critical role in ABT-263 plus sorafenib-induced apoptosis. Combining sorafenib with ABT-263 dramatically increased its efficacy in vivo.

CONCLUSION AND IMPLICATIONS

The anti-tumour activity of ABT-263 plus sorafenib may involve the induction of intrinsic cell apoptosis via inhibition of Akt, and reduced Bax and p21 expression. Our findings offer a novel effective therapeutic strategy for tumour treatment.     

Stimulatory effects of the multi-kinase inhibitor sorafenib on human bladder cancer cells

BACKGROUND AND PURPOSE

Sorafenib is an inhibitor of several intracellular signalling kinases with anti-proliferative, anti-angiogenic and pro-apoptotic effects in tumour cells. Sorafenib is used in the therapy of advanced renal cell carcinoma, and several phase II clinical trials are being carried out in patients with urothelial carcinomas.

EXPERIMENTAL APPROACH

Using a panel of human bladder cancer cell lines (RT4, T24, J82), we characterized systematically the effects of sorafenib on intracellular signalling, migration, proliferation and apoptosis.

KEY RESULTS

We demonstrated that at low concentrations (<1 µM), sorafenib is capable of significantly stimulating migration and proliferation of the bladder cancer cells. We hypothesize that these stimulatory effects on tumour cell functions might be explained by an activation of the Ras/ERK-1/2 signal transduction pathway. In addition, the comparison of different bladder cancer cell lines not only revealed a different biology (e.g. cell migration), but also a differential susceptibility to the anti-apoptotic effects of sorafenib. Finally, we confirmed in different bladder cancer cell lines the known inhibitory actions of sorafenib in pharmacological concentrations (≥3 µM) on ERK-1/2 phosphorylation, migration and proliferation, as well as the pro-apoptotic effects of the compound.

CONCLUSIONS AND IMPLICATIONS

Taken together, these findings suggest that although sorafenib has the potential to be used in the treatment of urothelial carcinoma, this compound might also activate bladder cancer cells at low concentrations. This should be relevant for dosing regiments to optimize the treatment with this promising anti-tumour drug.     

Sorafenib extends the survival time of patients with multiple recurrences of hepatocellular carcinoma after liver transplantation

Aim:

To determine the efficacy and toxicities of sorafenib in the treatment of patients with multiple recurrences of hepatocellular carcinoma (HCC) after liver transplantation in a Chinese population.

Methods:

Twenty patients with multiple recurrences of HCC after liver transplantation were retrospectively studied. They received either transarterial chemoembolization (TACE) or TACE combined with sorafenib.

Results:

The median survival times (MST) after multiple recurrences was 14 months (TACE+sorafenib group) and 6 months (TACE only group). The difference was significant in MST between the two groups (P=0.005). The TACE + sorafenib group had more stable disease (SD) patients than the TACE group. The most frequent adverse events of sorafenib were hand–foot skin reaction and diarrhea. In the univariate analysis, preoperative bilirubin and CHILD grade are found to be significantly associated with tumor-free survival time, the survival time after multiple recurrences and overall survival time. TACE+sorafenib group showed a better outcome than single TACE treatment group. In the multivariate COX regression modeling, the preoperative high CHILD grade was found to be a risk factor of tumor-free survival time. In addition, the preoperative high bilirubin grade was also found to be a risk factor of survival time after recurrence and overall survival time. Furthermore, survival time after recurrence and overall survival time were also associated with therapeutic schedule, which was indicated by the GROUP.

Conclusion:

Treatment with TACE and sorafenib is worthy of further study and may have more extensive application prospects.     

Bigelovin inhibits STAT3 signaling by inactivating JAK2 and induces apoptosis in human cancer cells

Aim:

To study the function and mechanism of bigelovin, a sesquiterpene lactone from the flower of Chinese herb Inula hupehensis, in regulating JAK2/STAT3 signaling and cancer cell growth.

Methods:

HepG2 cells stably transfected with the STAT3-responsive firefly luciferase reporter plasmid (HepG2/STAT3 cells), and a panel of human cancer cell lines were used to identify active compounds. Cell viability was measured using MTT assay. Western blotting was used to detect protein expression and phosphorylation. Kinase assays were performed and the reaction between bigelovin and thiol-containing compounds was analyzed with LC-MS.

Results:

Bigelovin (1–50 μmol/L) dose-dependently inhibited the IL-6-induced STAT3 activation in HepG2/STAT3 cells (IC₅₀=3.37 μmol/L) and the constitutive STAT3 activation in A549 and MDA-MB-468 cells. Furthermore, bigelovin dose-dependently inhibited JAK2 phosphorylation in HeLa and MDA-MB-468 cells, as well as the enzymatic activity of JAK2 in vitro (IC₅₀=44.24 μmol/L). Pretreatment of the cells with DTT (500 μmol/L) or GSH (500 μmol/L) eliminated the inhibitory effects of bigelovin on the IL-6-induced and the constitutive STAT3 activation. The results in LC-MS analysis suggested that bigelovin might react with cysteine residues of JAK2 leading to inactivation of JAK2. Bigelovin (5 and 20 μmol/L) had no effects on the signaling pathways of growth factors EGF, PDGF or insulin. Finally, bigelovin suppressed the cell viability and induced apoptosis in 10 different human cancer cell lines, particularly those with constitutively activated STAT3.

Conclusion:

Bigelovin potently inhibits STAT3 signaling by inactivating JAK2, and induces apoptosis of a variety of human cancer cells in vitro.     

Inhibition of hypoxia-inducible carbonic anhydrase-IX enhances hexokinase II inhibitor-induced hepatocellular carcinoma cell apoptosis

Aim:

The hypoxic condition within large or infiltrative hypovascular tumors produces intracellular acidification, which could activate many signaling pathways and augment cancer cell growth and invasion. Carbonic anhydrase-IX (CA-IX) is an enzyme lowering pH. This study is to examine whether hypoxia induces CA-IX in hepatocellular carcinoma (HCC) cells, and to evaluate its clinical implication in HCC patients.

Methods:

Human HCC cell lines (Huh-7 and HepG2 cells) were used, and cell growth was assessed using MTS assay. CA-IX expression and apoptotic/kinase signaling were evaluated using immunoblotting. The cells were transfected with CA-IX-specific siRNA, or treated with its inhibitor 4-(2-aminoethyl) benzenesulfonamide (CAI#1), and/or the hexokinase II inhibitor, 3-bromopyruvate (3-BP). A clinic pathological analysis of 69 patients who underwent an HCC resection was performed using a tissue array.

Results:

Incubation of HCC cells under hypoxia (1% O₂, 5% CO₂, 94% N₂) for 36 h significantly increased CA-IX expression level. CAI#1 (400 μmol/L) or CA-IX siRNA (100 μmol/L) did not influence HCC cell growth and induce apoptosis. However, CAI#1 or CA-IX siRNA at these concentrations enhanced the apoptosis induced by 3-BP (100 μmol/L). This enhancement was attributed to increased ER stress and JNK activation, as compared with 3-BP alone. Furthermore, a clinic pathological analysis of 69 HCC patients revealed that tumor CA-IX intensity was inversely related to E-cadherin intensity.

Conclusion:

Inhibition of hypoxia-induced CA-IX enhances hexokinase II inhibitor-induced HCC apoptosis. Furthermore, CA-IX expression profiles may have prognostic implications in HCC patients. Thus, the inhibition of CA-IX, in combination with a hexokinase II inhibitor, may be therapeutically useful in patients with HCCs that are aggressively growing in a hypoxic environment.     

Population pharmacokinetic analysis of sorafenib in patients with solid tumours

AIMS

To characterize the pharmacokinetics (PK) of sorafenib in patients with solid tumours and to evaluate the possible effects of demographic, clinical and pharmacogenetic (CYP3A4*1B, CYP3A5*3C, UGT1A9*3 and UGT1A9*5) covariates on the disposition of sorafenib.

METHODS

PK were assessed in 111 patients enrolled in five phase I and II clinical trials, where sorafenib 200 or 400 mg was administered twice daily as a single agent or in combination therapy. All patients were genotyped for polymorphisms in metabolic enzymes for sorafenib. Population PK analysis was performed by using nonlinear mixed effects modelling (NONMEM). The final model was validated using visual predictive checks and nonparametric bootstrap analysis.

RESULTS

A one compartment model with four transit absorption compartments and enterohepatic circulation (EHC) adequately described sorafenib disposition. Baseline bodyweight was a statistically significant covariate for distributional volume, accounting for 4% of inter-individual variability (IIV). PK model parameter estimates (range) for an 80 kg patient were clearance 8.13 l h⁻¹ (3.6–22.3 l h⁻¹), volume 213 l (50–1000 l), mean absorption transit time 1.98 h (0.5–13 h), fraction undergoing EHC 50% and average time to gall bladder emptying 6.13 h.

CONCLUSIONS

Overall, population PK analysis was consistent with known biopharmaceutical/PK characteristics of oral sorafenib. No clinically important PK covariates were identified.     

JAK2/STAT5/Bcl-xL signalling is essential for erythropoietin-mediated protection against apoptosis induced in PC12 cells by the amyloid β?peptide Aβ25–35

BACKGROUND AND PURPOSE

Erythropoietin (EPO) exerts neuroprotective actions in the CNS, including protection against apoptosis induced by the amyloid β−peptide Aβ_25–35. However, it remains unclear which signalling pathway activated by EPO is involved in this neuroprotection. Here, we have investigated whether JAK2/STAT5/Bcl-xL and ERK1/2 signalling pathways are essential for EPO-mediated protection against apoptosis induced by Aβ_25–35.

EXPERIMENTAL APPROACH

EPO was added to cultures of PC12 cells, 1 h before Aβ_25–35. For kinase inhibitor studies, AG490 and PD98059 were added to PC12 cells, 0.5 h before the addition of EPO. Transfection with siRNA was used to knockdown STAT5. Activation of JAK2/STAT5/Bcl-xL and ERK1/2 signalling pathways were investigated by Western blotting. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl-tetrazolium bromide assay and apoptosis was detected by TUNEL and acridine orange–ethidium bromide double staining.

KEY RESULTS

EPO increased phosphorylation of JAK2 and STAT5 in PC12 cells treated with Aβ_25–35. Furthermore, EPO modulated the nuclear translocation of phospho-STAT5, which increased expression of Bcl-xL and decreased levels of caspase-3. These beneficial effects were blocked by the JAK2 inhibitor, AG490 or STAT5 knockdown. However, the ERK1/2 pathway did not play a crucial role in our model.

CONCLUSIONS AND IMPLICATIONS

EPO protected PC12 cells against Aβ_25–35-induced neurotoxicity. Activation of JAK2/STAT5/Bcl-xL pathway was important in EPO-mediated neuroprotection. EPO may serve as a novel protective agent against Aβ_25–35-induced cytotoxicity in, for instance, Alzheimer''s disease.     

Inhibition of the STAT3 signaling pathway is involved in the antitumor activity of cepharanthine in SaOS2 cells

Aim:

To investigate the molecular mechanisms underlying the antitumor activity of cepharanthine (CEP), an alkaloid extracted from Stephania cepharantha Hayata.

Methods:

Human osteosarcoma cell line SaOS2 was used. MTT assay, Hoechst 33342 nuclear staining, flow cytometry, Western blotting and nude mouse xenografts of SaOS2 cells were applied to examine the antitumor activity of CEP in vitro and in vivo. The expression levels of STAT3 and its downstream signaling molecules were measured with Western blotting and immunochemistry analysis. The activity of STAT3 was detected based on the phosphorylation level of STAT3, luciferase gene reporter assay and translocation of STAT3 to the nucleus.

Results:

Treatment of SaOS2 cells with CEP (2.5–20 μmol/L) inhibited the cell growth in a concentration- and time-dependent manner. CEP (10 μmol/L) caused cell cycle arrest at G₁ phase and induced apoptosis of SaOS2 cells. CEP (10 and 15 μmol/L) significantly decreased the expression of STAT3 in SaOS2 cells. Furthermore, CEP (5 and 10 μmol/L) significantly inhibited the expression of target genes of STAT3, including the anti-apoptotic gene Bcl-xL and the cell cycle regulators c-Myc and cyclin D1. In nude mouse xenografts of SaOS2 cells, CEP (20 mg·kg⁻¹·d⁻¹, ip for 19 d) significantly reduced the volume and weight of the tumor.

Conclusion:

Our findings suggest that inhibition of STAT3 signaling pathway is involved in the anti-tumor activity of CEP.     

In rat renal fibroblasts, mycophenolic acid inhibits proliferation and production of the chemokine CCL2, stimulated by tumour necrosis factor-��

BACKGROUND AND PURPOSE

Renal fibroblasts play a pivotal role in the development of tubulointerstitial fibrosis, a condition highly predictive of progression towards end-stage renal disease. The present study investigated the anti-mitogenic and anti-inflammatory effects of an inhibitor of inosine monophosphate dehydrogenase, mycophenolic acid (MPA) and the mechanisms underlying its action in normal rat kidney fibroblasts (49F cells).

EXPERIMENTAL APPROACH

Proliferation of 49F cells was studied by tetrazole 3-(4, 5-dimethylthiazol-2-yl-)-2,5-diphenyltetrazolium bromide (MTT) test, bromodeoxyuridine incorporation and flow cytometry. The cyclins, tumour suppressor genes and phospho-mitogen-activated protein kinases (MAPKs) were semiquantified by immunoblotting. Apoptosis was measured by quantifying the fragmented DNA and the activity of caspase 3. The monocyte chemokine CCL2 was measured by ELISA. The mRNA expression of CCL2 was measured by real-time PCR.

KEY RESULTS

Mycophenolic acid dose-dependently inhibited steady-state proliferation of 49F cells by up-regulation of p21, p27 and p53, in association with a decrease in cyclins D2 and E. Treatment with MPA also triggered apoptosis of 49F cells by activating the caspase 3 cascade. Furthermore, MPA attenuated tumour necrosis factor-α-induced CCL2 expression through down-regulation of p38 MAPK, but not that of ERK1/2 or JNK.

CONCLUSIONS AND IMPLICATIONS

The anti-mitogenic and anti-inflammatory effects of MPA were mediated by up-regulation of cell cycle inhibitors and pro-apoptotic signals, and by suppression of p38 MAPK pathway respectively. This dual effect of MPA may form the rationale for animal or clinical trials for the treatment of fibrotic renal diseases.