Evaluation of the liver and blood micronucleus, and comet assay end points in a 14-day repeated-dose study with methyl carbamate and 1,3-propane sultone.

The repeated-dose liver micronucleus (RDLMN) assay is a novel method for detecting genotoxic chemicals. Two carcinogens methyl carbamate (MC) and 1,3-propane sultone (PS) were evaluated for the liver micronucleus in a 14-day repeated-dose study with Crl: CD (SD) IGS rats. Additionally, micronucleated reticulocytes (MN-RET) in peripheral blood and DNA damage (alkaline comet assay) in the liver were also assessed in the same animals. Ten groups of five male Crl: CD (SD) IGS rats were treated once daily with MC (300, 600 or 1200 mg/kg/day), PS (37.5, 75 or 150 mg/kg/day), negative control or three positive controls by oral gavage for 15 days. Blood samples were collected at 3 h after the last administration for determining MN-RET frequencies (%MN-RET), and the livers were sampled for determining the frequency of micronuclei and DNA damage. MC was negative in the comet assay, liver micronucleus assay and reticulocyte micronucleus assay, while PS was positive in all three assays. These results are consistent with the previous genotoxic findings of MC and PS. Therefore, the liver micronucleus assay can be effectively integrated into repeated-dose studies in animals. Moreover, integration of multiple genotoxicity end points into one study can reduce the number of animals, boost the experimental efficiency, and provides a comprehensive evaluation of the genotoxic potential of chemicals.

Discovery of a Candidate Containing an (S)-3,3-Difluoro-1-(4-methylpiperazin-1-yl)-2,3-dihydro-1H-inden Scaffold as a Highly Potent Pan-Inhibitor of the BCR-ABL Kinase Including the T315I-Resistant Mutant for the Treatment of Chronic Myeloid Leukemia.

BCR-ABL kinase inhibition is an effective strategy for the treatment of chronic myeloid leukemia (CML). Herein, we report compound 3a-P1, bearing a difluoro-indene scaffold, as a novel potent pan-inhibitor against BCR-ABL mutants, including the most refractory T315I mutant. As the privileged (S)-isomer compared to its (R)-isomer 3a-P2, 3a-P1 exhibited potent antiproliferative activities against K562 and Ku812 CML cells and BCR-ABL and BCR-ABLT315I BaF3 cells, with IC50 values of 0.4, 0.1, 2.1, and 4.7 nM, respectively. 3a-P1 displayed a good safety profile in a battery of assays, including single-dose toxicity, hERG K+, and genotoxicity. It also showed favorable mice pharmacokinetic properties with a good oral bioavailability (32%), a reasonable half-life (4.61 h), and a high exposure (1386 h·ng/mL). Importantly, 3a-P1 demonstrated a higher potency than ponatinib in a mice xenograft model of BaF3 harboring BCR-ABLT315I. Overall, the results indicate that 3a-P1 is a promising drug candidate for the treatment of CML to overcome the imatinib-resistant T315I BCR-ABL mutation.

Tetrandrine is a potent cell autophagy agonist via activated intracellular reactive oxygen species.

BACKGROUND: Autophagy is an evolutionarily conserved cellular process that involves the lysosomal degradation of proteins and organelles and the recycling of cellular components to ensure cellular survival under external or internal stress. Numerous data has indicated that autophagy can be successfully targeted for the treatment of multiple cancers. We have previously demonstrated that tetrandrine, a bisbenzylisoquinoline alkaloid isolated from the broadly used Chinese medicinal herb Stephaniae tetrandrae, exhibits potent antitumor effects when used either alone or in combination with other drugs. RESULTS: In the present study, we showed that tetrandrine is a broad-spectrum potent autophagy agonist. Although low-dose tetrandrine treatment does not affect cell viability, it can potently induce autophagy in a variety of cell lines, including cancerous cells and nontumorigenic cells. The autophagy inhibitors 3-methyladenine (3-MA) and chloroquine (CQ), effectively blocked tetrandrine-induced autophagy. Moreover, tetrandrine significantly triggered the induction of mitophagy. The underlying mechanisms are associated with the tetrandrine-induced production of intracellular reactive oxygen species (ROS), which plays a critical role in tetrandrine-induced autophagy. CONCLUSIONS: Here, we report that tetrandrine is a potent cell autophagy agonist and may have a wide range of applications in the fields of antitumor therapy and basic scientific research.

Schisandrin B protects PC12 cells against oxidative stress of neurodegenerative diseases.

Increasing evidence places Schisandrin B (Sch B) at an important position in nerve protection, indicating that Sch B might play a positive role in the therapy of neurodegenerative diseases. However, there is little information on it. Our studies showed that pretreatment with Sch B could reduce lactate dehydrogenase, malondialdehyde, and reactive oxygen species release and significantly increase the cell viability and the superoxide dismutase level. Sch B (10 µM) markedly inhibited cell apoptosis, whereas LY294002 (20 µM), a phosphatidylinositol-3 kinase inhibitor, blocked the antiapoptotic effect. More importantly, Sch B (10 µM) increased the phosphoprotein kinase B/protein kinase B (Akt) and B-cell lymphoma-2/Bcl-2 associated X protein ratios on preincubation with cells for 2 h, which was then inhibited by LY294002 (20 µM). Results indicate that Sch B can protect PC12 cells from apoptosis by activating the phosphatidylinositol-3 kinase/Akt signaling pathway and may emerge as a potential drug for neurodegenerative diseases.

Tetrandrine induces autophagy and differentiation by activating ROS and Notch1 signaling in leukemia cells.

All-trans retinoic acid (ATRA) is a differentiating agent for the treatment of acute promyelocytic leukemia (APL). However, the therapeutic efficacy of ATRA has limitations. Tetrandrine is a traditional Chinese medicinal herb extract with antitumor effects. In this study, we investigated the effects of tetrandrine on human PML-RARα-positive acute promyelocytic leukemia cells. Tetrandrine inhibited tumors in vivo. It induced autophagy and differentiation by triggering ROS generation and activating Notch1 signaling. Tetrandrine induced autophagy and differentiation in M5 type patient primary leukemia cells. The in vivo results indicated that low concentrations of tetrandrine inhibited leukemia cells proliferation and induced autophagy and then facilitated their differentiation, by activating ROS and Notch1 signaling. We suggest that tetrandrine is a potential agent for the treatment of APL by inducing differentiation of leukemia cells.

Synergistic anti-tumour effects of tetrandrine and chloroquine combination therapy in human cancer: a potential antagonistic role for p21.

BACKGROUND AND PURPOSE: Tetrandrine, a bisbenzylisoquinoline alkaloid isolated from the Chinese medicinal herb Stephaniae tetrandrae, has a long history in Chinese clinical applications to treat diverse diseases. Tetrandrine induced apoptosis or, at low concentrations, autophagy of human hepatocellular carcinoma cells. Here we have tested the effects of inhibitors of autophagy such as chloroquine, on the response to low concentrations of tetrandrine in cancer cells. EXPERIMENTAL APPROACH: Cultures of several cancer cell lines, including Huh7, U251, HCT116 and A549 cells, were exposed to tetrandrine, chloroquine or a combination of these compounds. Cell viability and content of reactive oxygen species (ROS) were measured and synergy assessed by calculation of the combination index. Western blot and RT-PCR assays were also used along with fluorescence microscopy and histochemical techniques. KEY RESULTS: Combinations of tetrandrine and chloroquine were more cytotoxic than the same concentrations used separately and these effects showed synergy. Such effects involved increased ROS generation and were dependent on caspase-3 but independent of Akt activity. Blockade of tetrandrine-induced autophagy with 3-methyladenine or bafilomycin-A1 induced apoptosis in cancer cells. Lack of p21 protein (p21(-/-) HCT116 cells) increased sensitivity to the apoptotic effects of the combination of tetrandrine and chloroquine. In a tumour xenograft model in mice, combined treatment with tetrandrine and chloroquine induced ROS accumulation and cell apoptosis, and decreased tumour growth. CONCLUSIONS AND IMPLICATIONS: The combinations of tetrandrine and chloroquine exhibited synergistic anti-tumour activity, in vitro and in vivo. Our results suggest a novel therapeutic strategy for tumour treatment.

[Effect of Schisandra chinensis lignans on neuronal apoptosis and p-AKT expression of rats in cerebral ischemia injury model].

OBJECTIVE: To observe the effect of Schisandra chinensis lignans (SCL) on neuronal apoptosis and PI3K/AKT signaling pathway of rats in the cerebral ischemia injury model, and study its possible mechanism. METHOD: Rats were orally administered SCL high, middle and low dose groups (100, 50, 25 mg x kg(-1)) for 14 days. The cerebral ischemia injury model was established by using the suture-occluded method to rate the neurological functions. The cerebral infarction area was observed by TTC staining. The pathological changes in brain tissues were determined by HE staining. Bcl-2 and Bax expressions were detected by immunohistochemical assay. The protein expressions of p-AKT and AKT were assayed by Western blotting. RESULT: Compared with the model group, SCL high, middle and low dose groups showed reduction in the cerebral infarction area to varying degrees, improve the pathological changes in brain tissues, promote the expression of apoptin Bcl-2 and p-AKT, and inhibit the expression of apoptin Bax. CONCLUSION: SCL shows a protective effect on rats with cerebral ischemia injury. Its mechanism may be related to the increase in p-AKT ability and antiischemic brain injury capacity and the inhibition of nerve cells.

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.

CBP loss cooperates with PTEN haploinsufficiency to drive prostate cancer: implications for epigenetic therapy.

Despite the high incidence and mortality of prostate cancer, the etiology of this disease is not fully understood. In this study, we develop functional evidence for CBP and PTEN interaction in prostate cancer based on findings of their correlate expression in the human disease. Cbp(pc-/-);Pten(pc+/-) mice exhibited higher cell proliferation in the prostate and an early onset of high-grade prostatic intraepithelial neoplasia. Levels of EZH2 methyltransferase were increased along with its Thr350 phosphorylation in both mouse Cbp(-/-); Pten(+/-) and human prostate cancer cells. CBP loss and PTEN deficiency cooperated to trigger a switch from K27-acetylated histone H3 to K27-trimethylated bulk histones in a manner associated with decreased expression of the growth inhibitory EZH2 target genes DAB2IP, p27(KIP1), and p21(CIP1). Conversely, treatment with the histone deacetylase inhibitor panobinostat reversed this switch, in a manner associated with tumor suppression in Cbp(pc-/-);Pten(pc+/-) mice. Our findings show how CBP and PTEN interact to mediate tumor suppression in the prostate, establishing a central role for histone modification in the etiology of prostate cancer and providing a rationale for clinical evaluation of epigenetic-targeted therapy in patients with prostate cancer.

Apigenin sensitizes colon cancer cells to antitumor activity of ABT-263.

Apigenin is an edible plant-derived flavonoid that shows modest antitumor activities in vitro and in vivo. Apigenin treatment resulted in cell growth arrest and apoptosis in various types of tumors by modulating several signaling pathways. In the present study, we evaluated interactions between apigenin and ABT-263 in colon cancer cells. We observed a synergistic effect between apigenin and ABT-263 on apoptosis of colon cancer cells. ABT-263 alone induced limited cell death while upregulating expression of Mcl-1, a potential mechanism for the acquired resistance to ABT-263. The presence of apigenin antagonized ABT-263-induced Mcl-1 upregulation and dramatically enhanced ABT-263-induced cell death. Meanwhile, apigenin suppressed AKT and ERK activation. Inactivation of either AKT or ERK by lentivirus-transduced shRNA or treatment with specific small-molecule inhibitors of these pathways enhanced ABT-263-induced cell death, mirroring the effect of apigenin. Moreover, the combination response was associated with upregulation of Bim and activation of Bax. Downregulation of Bax eliminated the synergistic effect of apigenin and ABT-263 on cell death. Xenograft studies in SCID mice showed that the combined treatment with apigenin and ABT-263 inhibited tumor growth by up to 70% without obvious adverse effects, while either agent only inhibited around 30%. Our results demonstrate a novel strategy to enhance ABT-263-induced antitumor activity in human colon cancer cells by apigenin via inhibition of the Mcl-1, AKT, and ERK prosurvival regulators.

p62/sequestosome-1 up-regulation promotes ABT-263-induced caspase-8 aggregation/activation on the autophagosome.

Autophagy and apoptosis regulate cancer cell viability in response to cytotoxic stress; however, their functional relationship remains unclear. p62/sequestosome 1 is a multifunctional protein and a signaling hub that shuttles ubiquitinated proteins to the lysosome during autophagy. Autophagy inhibition up-regulates p62, and prior data suggest that p62 may mediate apoptosis. Here, we demonstrate that p62 can regulate a caspase-8-dependent apoptosis in response to the BH3 mimetic agent, ABT-263. Up-regulation of p62 was shown to enhance ABT-263-induced caspase-8 activation that was Bax-dependent and resulted from mitochondrial amplification. Dependence upon caspase-8 was confirmed using caspase-8-deficient cells and by caspase-8 siRNA. Ectopic wild-type p62, but not p62 mutants with loss of ability to promote apoptosis, was shown to co-localize with caspase-8 and to promote its self-aggregation in ABT-263-treated cells, shown using a bimolecular fluorescence complementation assay. Endogenous p62 co-localized with caspase-8 in the presence of ABT-263 plus an autophagy inhibitor. Caspase-8 was shown to interact and co-localize with the autophagosome marker, LC3II. Knockdown of p62 attenuated binding between caspase-8 and LC3II, whereas p62 overexpression enhanced the co-localization of caspase-8 aggregates with LC3. LC3 knockdown did not affect interaction between caspase-8 and p62, suggesting that p62 may facilitate caspase-8 translocation to the autophagosomal membrane. A direct activator of caspase-8, i.e., TRAIL, alone or combined with ABT-263, induced caspase-8 aggregation and co-localization with p62 that was associated with a synergistic drug interaction. Together, these results demonstrate that up-regulation of p62 can mediate apoptosis via caspase-8 in the setting of autophagy inhibition.

Dual targeting of mTORC1/C2 complexes enhances histone deacetylase inhibitor-mediated anti-tumor efficacy in primary HCC cancer in vitro and in vivo.

BACKGROUND & AIMS: The mammalian target of rapamycin (mTOR) plays a pivotal role in hepatocellular carcinoma (HCC). Previous studies indicated that inhibition of mTORC1 enhanced histone deacetylase inhibitors (HDACis)-mediated anti-tumor activity, accompanied with feedback activation of AKT. Therefore, dual targeting of mTORC1/C2 should be more efficient in suppressing AKT activity and in enhancing the anti-tumor activity of HDACi in HCC. METHODS: The interactions between mTOR kinase inhibitors (mTORKis) (i.e., Pp242, AZD8055, OSI027) and HDACis (i.e., SAHA, LBH589) were examined in vitro using HCC cell lines and in vivo using patient-derived primary HCC xenografts on SCID mice. RESULTS: mTORKis significantly enhanced HDACi-induced apoptosis in HCC cells. The inhibition of both mTORC1/2 not only efficiently blocked mTORC1 signaling, but also abrogated AKT-feedback activation caused by selective mTORC1 inhibition. The co-treatment of mTORKi and HDACi further inhibited AKT signaling and upregulated Bim. Dysfunction of mTORC2 by shRNA significantly lowered the threshold of HDACi-induced cytotoxicity by abrogating AKT activation. Knockdown of AKT1 sensitized Pp242/HDACi-induced apoptosis and ectopic expression of constitutively active AKT1 abrogated the combination-induced cytotoxicity, indicating AKT plays a vital role in the combination-induced effects. Knockdown of Bim prevented Pp242/HDACis-induced cytotoxicity in HCC. Lastly, in vivo studies indicated that the combination of AZD8055 and SAHA almost completely inhibited tumor-growth, without obvious adverse effects, by abrogating AKT and upregulating Bim; while either agent alone shows only 30% inhibition in primary HCC xenografts. CONCLUSIONS: Our findings suggest that a combining-regimen of mTORKi and HDACi may be an effective therapeutic strategy for HCC.

Phase II trial of vorinostat in combination with bortezomib in recurrent glioblastoma: a north central cancer treatment group study.

Vorinostat, a histone deacetylase (HDAC) inhibitor, has shown evidence of single-agent activity in glioblastoma (GBM), and in preclinical studies, we have demonstrated significant synergistic cytotoxicity between HDAC inhibitors and proteasome inhibitors in GBM cell lines. We therefore conducted a phase II trial to evaluate the efficacy of vorinostat in combination with the proteasome inhibitor bortezomib in patients with recurrent GBM. Vorinostat was administered at a dose of 400 mg daily for 14 days of a 21-day cycle, and bortezomib was administered at a dose of 1.3 mg/m(2) intravenously on days 1, 4, 8, and 11 of the cycle. A total of 37 patients were treated, and treatment was well tolerated: grade 3, 4 nonhematologic toxicity occurred in 30% of patients and consisted mainly of fatigue (14%) and neuropathy (5%); grade 3, 4 hematologic toxicity occurred in 37% of patients and consisted of thrombocytopenia (30%), lymphopenia (4%), and neutropenia (4%). The trial was closed at the predetermined interim analysis, with 0 of 34 patients being progression-free at 6 months. One patient achieved a partial response according to the Macdonald criteria. The median time to progression for all patients was 1.5 months (range, 0.5-5.6 months), and median overall survival (OS) was 3.2 months. Patients who had received prior bevacizumab therapy had a shorter time to progression and OS, compared with those who had not. On the basis of the results of this phase II study, further evaluation of the vorinostat-bortezomib combination in GBM patients in this dose and schedule is not recommended.

Inhibition of mTOR kinase by AZD8055 can antagonize chemotherapy-induced cell death through autophagy induction and down-regulation of p62/sequestosome 1.

AZD8055 is an ATP-competitive inhibitor of mammalian target of rapamycin (mTOR) that forms two multiprotein complexes, mTORC1 and mTORC2, and negatively regulates autophagy. We demonstrate that AZD8055 stimulates and potentiates chemotherapy-mediated autophagy, as shown by LC3I-II conversion and down-regulation of the ubiquitin-binding protein p62/sequestosome 1. AZD8055-induced autophagy was pro-survival as shown by its ability to attenuate cell death and DNA damage (p-H2AX), and to enhance clonogenic survival by cytotoxic chemotherapy. Autophagy inhibition by siRNA against Beclin 1 or LC3B, or by chloroquine, partially reversed the cytoprotective effect of AZD8055 that was independent of cell cycle inhibition. The pro-survival role of autophagy was confirmed using ectopic expression of Beclin 1 that conferred cytoprotection. To determine whether autophagy-mediated down-regulation of p62/sequestosome 1 contributes to its pro-survival role, we generated p62 knockdown cells using shRNA that showed protection from chemotherapy-induced cell death and DNA damage. We also overexpressed wild-type (wt) p62 that promoted chemotherapy-induced cell death, whereas mutated p62 at functional domains (PB1, UBA) failed to do so. The ability of ectopic wt p62 to promote cell death was blocked by AZD8055. AZD8055 was shown to inhibit phosphorylation of the autophagy-initiating kinase ULK1 at Ser(757) and inhibited known targets of mTORC1 (p-mTOR Ser(2448), p70S6K, p-S6, p4EBP1) and mTORC2 (p-mTOR Ser(2481), p-AKT Ser(473)). Knockdown of mTOR, but not Raptor or Rictor, reduced p-ULK1 at Ser(757) and enhanced chemotherapy-induced autophagy that resulted in a similar cytoprotective effect as shown for AZD8055. In conclusion, AZD8055 inhibits mTOR kinase and ULK1 phosphorylation to induce autophagy whose pro-survival effect is due, in part, to down-regulation of p62.

Mcl-1 downregulation by YM155 contributes to its synergistic anti-tumor activities with ABT-263.

YM155, a small-molecule survivin suppressant, exhibits anti-tumor activities in vitro, in vivo and in clinical trials. However, the mechanism of YM155 action remains unclear. In this study, YM155 was administered to a panel of cell lines and the effects of YM155 on Bcl-2 family members were analyzed. Our results show that YM155 strikingly downregulates Mcl-1 in a broad spectrum of cancer cell lines and that the Mcl-1 modulation occurs at the transcriptional level, independently of survivin modulation or caspase activity. Furthermore, analysis of the contribution of Mcl-1 or survivin downregulation to YM155-induced cell death in vitro showed that knockdown of Mcl-1 sensitizes cells to YM155-induced cytotoxicity. Finally, our data demonstrate that downregulation of Mcl-1 by YM155 synergistically lowers the threshold of Bcl-2 family member inhibitor ABT-263-induced cell death. Our findings reveal a novel mechanism by which survivin-independent Mcl-1 suppression plays a critical role in YM155-mediated anti-tumor activities. YM155 treatment in combination with ABT-263 thus affords a new strategy for cancer treatment.

Improved response to nab-paclitaxel compared with cremophor-solubilized paclitaxel is independent of secreted protein acidic and rich in cysteine expression in non-small cell lung cancer.

BACKGROUND: The secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein that is produced by tumor and/or neighboring stroma. SPARC expression is thought to facilitate the intracellular accumulation of nanoparticle albumin-bound paclitaxel (nab-paclitaxel, abraxane [ABX]). Gene hypermethylation is a common mechanism for loss of SPARC expression in non-small cell lung cancer (NSCLC). We aim to demonstrate the role of SPARC expression as biomarker for treatment selection using ABX in NSCLC and to evaluate the presence of synergistic antitumor effect when a demethylating agent is combined with ABX. METHODS: We analyzed the SPARC messenger RNA expression and SPARC gene methylation status in 13 NSCLC cell lines and 22 minimally passaged patient-derived (PD) NSCLC tumors using real-time (RT) polymerase chain reaction. The effect of ABX on tumor growth was compared with cremophor-solubilized paclitaxel (taxol) in severe combined immunodeficiency mice bearing SPARC-positive PD xenografts. The effect of pretreatment with a demethylating agent, 5-Aza-2'-deoxycytidine (DEC) in SPARC-negative tumors was assessed. RESULTS: SPARC expression was weak to absent in 62% of established NSCLC cell lines and 68% of PD NSCLC tumor xenografts. SPARC expression could be up-regulated/restored by DEC treatment in both SPARC-negative cell lines and PD xenografts in vitro and in vivo. ABX demonstrated better antitumor efficacy than equitoxic dose of taxol in SPARC-expressing xenografts and some SPARC-negative xenografts. At equimolar doses in vitro, there was similar increased cytotoxicity on DEC pretreatment with either ABX or taxol in SPARC-negative cell lines. At equitoxic doses, there was similar additive antitumor activity of DEC with either ABX or taxol in SPARC-negative PD xenografts. CONCLUSION: Endogenous SPARC status is somewhat uncorrelated with response to ABX in NSCLC. The greater antitumor effect of ABX compared with equitoxic dose of taxol observed in SPARC-expressing NSCLC tumors can also be seen in some SPARC-negative tumors. DEC pretreatment similarly enhanced antitumor activity with either ABX or taxol in SPARC-negative tumors.

Sulfatase 2 protects hepatocellular carcinoma cells against apoptosis induced by the PI3K inhibitor LY294002 and ERK and JNK kinase inhibitors.

BACKGROUND: Sulfatase 2 (SULF2), an extracellular heparan sulphate 6-O-endosulphatase, has an oncogenic effect in hepatocellular carcinoma (HCC) that is partially mediated through glypican 3, which promotes heparin-binding growth factor signalling and HCC cell growth. SULF2 also increases phosphorylation of the anti-apoptotic Akt kinase substrate GSK3ß and SULF2 expression is associated with a decreased apoptotic index in human HCCs. METHODS: We investigated the functional and mechanistic effects of SULF2 on drug-induced apoptosis of HCC cells using immunohistochemistry, Western immunoblotting, gene transfection, real-time quantitative polymerase chain reaction, MTT and apoptosis assays and immunocytochemistry. RESULTS: The increased expression of SULF2 in human HCCs was confirmed by immunohistochemistry and immunoblotting. Treatment with inhibitors of MEK, JNK and PI3 kinases decreased the viability of SULF2-negative Hep3B HCC cells and induced apoptotic caspase 3 and 7 activity, which was most strongly induced by the PI3K inhibitor LY294002. Forced expression of SULF2 in Hep3B cells significantly decreased activity of the apoptotic caspases 3 and 7 and induced resistance to LY294002-induced apoptosis. As expected, LY294002 inhibited activation of Akt kinase by PI3K. Conversely, knockdown of SULF2 using an shRNA construct targeting the SULF2 mRNA induced profound cell growth arrest and sensitized the endogenously SULF2-expressing HCC cell lines Huh7 and SNU182 to drug-induced apoptosis. The effects of knockdown of SULF2 on HCC cells were mediated by decreased Akt phosphorylation, downregulation of cyclin D1 and the anti-apoptotic molecule Bcl-2, and upregulation of the pro-apoptotic molecule BAD. CONCLUSION: The prosurvival, anti-apoptotic effect of SULF2 in HCC is mediated through activation of the PI3K/Akt pathway.

The oncogenic effect of sulfatase 2 in human hepatocellular carcinoma is mediated in part by glypican 3-dependent Wnt activation.

UNLABELLED: Heparan sulfate proteoglycans (HSPGs) act as coreceptors or storage sites for growth factors and cytokines such as fibroblast growth factor and Wnts. Glypican 3 (GPC3) is the most highly expressed HSPG in hepatocellular carcinoma (HCC). Sulfatase 2 (SULF2), an enzyme with 6-O-desulfatase activity on HSPGs, is up-regulated in 60% of primary HCCs and is associated with a worse prognosis. We have previously shown that the oncogenic effect of SULF2 in HCC may be mediated in part through up-regulation of GPC3. Here we demonstrate that GPC3 stimulates the Wnt/ß-catenin pathway and mediates the oncogenic function of SULF2 in HCC. Wnt signaling in vitro and in vivo was assessed in SULF2-negative Hep3B HCC cells transfected with SULF2 and in SULF2-expressing Huh7 cells transfected with short hairpin RNA targeting SULF2. The interaction between GPC3, SULF2, and Wnt3a was assessed by coimmunoprecipitation and flow cytometry. ß-catenin-dependent transcriptional activity was assessed with the TOPFLASH (T cell factor reporter plasmid) luciferase assay. In HCC cells, SULF2 increased cell surface GPC3 and Wnt3a expression, stabilized ß-catenin, and activated T cell factor transcription factor activity and expression of the Wnt/ß-catenin target gene cyclin D1. Opposite effects were observed in SULF2-knockdown models. In vivo, nude mouse xenografts established from SULF2-transfected Hep3B cells showed enhanced GPC3, Wnt3a, and ß-catenin levels. CONCLUSION: Together, these findings identify a novel mechanism mediating the oncogenic function of SULF2 in HCC that includes GPC3-mediated activation of Wnt signaling via the Wnt3a/glycogen synthase kinase 3 beta axis.

HER receptor signaling confers resistance to the insulin-like growth factor-I receptor inhibitor, BMS-536924.

We have reported previously the activity of the insulin-like growth factor-I (IGF-IR)/insulin receptor (InsR) inhibitor, BMS-554417, in breast and ovarian cancer cell lines. Further studies indicated treatment of OV202 ovarian cancer cells with BMS-554417 increased phosphorylation of HER-2. In addition, treatment with the pan-HER inhibitor, BMS-599626, resulted in increased phosphorylation of IGF-IR, suggesting a reciprocal cross-talk mechanism. In a panel of five ovarian cancer cell lines, simultaneous treatment with the IGF-IR/InsR inhibitor, BMS-536924 and BMS-599626, resulted in a synergistic antiproliferative effect. Furthermore, combination therapy decreased AKT and extracellular signal-regulated kinase activation and increased biochemical and nuclear morphologic changes consistent with apoptosis compared with either agent alone. In response to treatment with BMS-536924, increased expression and activation of various members of the HER family of receptors were seen in all five ovarian cancer cell lines, suggesting that inhibition of IGF-IR/InsR results in adaptive up-regulation of the HER pathway. Using MCF-7 breast cancer cell variants that overexpressed HER-1 or HER-2, we then tested the hypothesis that HER receptor expression is sufficient to confer resistance to IGF-IR-targeted therapy. In the presence of activating ligands epidermal growth factor or heregulin, respectively, MCF-7 cells expressing HER-1 or HER-2 were resistant to BMS-536924 as determined in a proliferation and clonogenic assay. These data suggested that simultaneous treatment with inhibitors of the IGF-I and HER family of receptors may be an effective strategy for clinical investigations of IGF-IR inhibitors in breast and ovarian cancer and that targeting HER-1 and HER-2 may overcome clinical resistance to IGF-IR inhibitors.

BRAF V600E disrupts AZD6244-induced abrogation of negative feedback pathways between extracellular signal-regulated kinase and Raf proteins.

AZD6244 (ARRY 142886) is a potent and selective mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor currently in early clinical trials. We examined the activity of AZD6244 in a panel of non-small cell lung cancer and a panel of cell lines representing many cancer types using in vitro growth assays. AZD6244 induced G(0)-G(1) cell cycle arrest in sensitive cell lines that primarily included cells containing the BRAF V600E mutation. In these cells, G(0)-G(1) arrest is accompanied by the up-regulation of the cell cycle inhibitors p21(WAF1) and p27(Kip1) and down-regulation of cyclin D1. In the majority of cell lines tested, including those with K-ras or non-V600E BRAF mutations, AZD6244 induced the accumulation of phospho-MEK, an effect not observed in the most sensitive BRAF V600E-containing cells. Accumulation of phospho-MEK in non-V600E-containing cell lines is due to abrogation of negative feedback pathways. BRAF V600E disrupts negative feedback signaling, which results in enhanced baseline phospho-MEK expression. Exogenous expression of BRAF V600E disrupts feedback inhibition but does not sensitize cells to AZD6244. Specific suppression of endogenous BRAF V600E does not confer resistance to AZD6244 but enhances sensitivity to AZD6244. Thus, our findings show that BRAF V600E marks cells with an in vitro requirement for MAPK signaling to support proliferation. These cells are exquisitely sensitive to AZD6244 (IC(50), <100 nmol/L), have high baseline levels of phospho-MEK, and lack feedback inhibition between ERK and Raf. These data suggest an approach to identifying cells that may be sensitive to AZD6244 and other MEK inhibitors.