- Email: [email protected]
Sorafenib and triptolide as combination therapy for hepatocellular carcinoma
Sorafenib and triptolide as combination therapy for hepatocellular carcinoma Osama A. Alsaied, MD,a Veena Sangwan, PhD,b Sulagna Banerjee, PhD,b Tara C. Krosch, MD,a Rohit Chugh, MBBS,b Ashok Saluja, MD,b Selwyn M. Vickers, MD,c and Eric H. Jensen, MD,d Minneapolis, MN, and Birmingham, AL
Introduction. Sorafenib is the only drug approved by the Food and Drug Administration for metastatic hepatocellular carcinoma (HCC). Triptolide, a diterpene triepoxide, exhibits antineoplastic properties in multiple tumor cell types. In this study, we examined the effects of these agents and their combination on HCC in vitro and in vivo models. Methods. HuH-7 and PLC/PRF/5 cells were treated with triptolide (50 nM), sorafenib (1.25 or 2.5 mM), or a combination of both. Cell viability assay (CCK-8), caspase 3&7 activation, and nuclear factor kB assays were performed. For in vivo studies, 40 mice were implanted with subcutaneous HuH7 tumors and divided into four treatment groups (n = 10); saline control, sorafenib 10 mg/kg PO daily (S), Minnelide (a prodrug of triptolide) 0.21 mg/kg intraperitoneally7 daily (M), and combination of both (C). Tumor volumes were assessed weekly. Results. The combination of triptolide and sorafenib was superior to either drug alone in inducing apoptosis and decreasing viability, whereas triptolide alone was sufficient to decrease nuclear factor kB activity. After 2 weeks of treatment, tumor growth inhibition rates were S = 59%, M = 84%, and C = 93%, whereas tumor volumes in control animals increased by 9-fold. When crossed over to combination treatment, control mice tumor growth volumes plateaued over the following 4 weeks. Conclusion. The combination of sorafenib and triptolide is superior to single drug treatment in increasing cell death and apoptosis in vitro. Combining sorafenib with Minnelide inhibited tumor growth with greater efficacy than single-agent treatments. Importantly, in vivo combination treatment allowed for using a lesser dose of sorafenib (10 mg/kg), which is less than 10% of currently prescribed dose for HCC patients. Therefore, combination treatment could have translational potential in the management of HCC. (Surgery 2014;156:270-9.) From the Division of General Surgery,a Division of Basic and Translational Research,b and Division of Surgical Oncology, Department of Surgery, d University of Minnesota, Minneapolis, MN; and School of Medicine,c University of Alabama, Birmingham, AL
HEPATOCELLULAR CARCINOMA (HCC) is currently the sixth most-common cancer and is the thirdleading cause of cancer-related deaths worldwide.1,2 Chronic liver disease secondary to viral hepatitis is responsible for the majority of HCC Dr Saluja has a significant interest in and is the Chief Scientific Officer and a consultant for Minneamrita, a company that may commercially benefit from the results of this research. This relationship has been reviewed and managed by the University of Minnesota in accordance with its conflict of interest policies. Presented at the 9th Annual Academic Surgical Congress in San Diego, CA, February 4–6, 2014. Accepted for publication April 28, 2014. Reprint requests: Eric H. Jensen, MD, Division of Surgical Oncology, University of Minnesota Medical School, Minneapolis, MN 55455. E-mail: [email protected] 0039-6060/$ - see front matter Ó 2014 Mosby, Inc. All rights reserved. http://dx.doi.org/10.1016/j.surg.2014.04.055
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cases.1 In the United States, the incidence of HCC is related closely to hepatitis C infection and has been increasing during the past four decades.3 The prognosis of HCC is very poor with a 5-year survival for all stages at 15%, but with successful resection or liver transplantation, 5-year survival increases to 50% and 70%, respectively.4 Nonetheless, curative operative therapy is only offered to 1530% of all patients because of the advanced nature of the disease at the time of diagnosis.5,6 In 2007, the Sorafenib HCC Assessment Randomized Protocol Trial (SHARP) trial showed promising results with sorafenib, a multikinase inhibitor drug that increases median survival of patients with advanced HCC.5 Sorafenib targets a wide variety of growth factor and angiogenesis receptors, such as vascular endothelial growth factor receptor (VEGFR)2, VEGFR3, platelet-derived
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growth factor receptor, and c-KIT and affects multiple signal transduction pathways.7-10 The mechanism of action of sorafenib remains unclear. Minnelide is a water-soluble prodrug of triptolide, a diterpene triepoxide, derived from the Chinese herb Tripterygium wilfordii. Triptolide has been used in China to treat inflammatory conditions and cancers. Our group is currently investigating Minnelide in a phase 1 clinical trial for its antitumoral activity in gastrointestinal cancers.11-13 METHODS Cell culture. HuH-7 hepatocellular carcinoma cells (p53 mutant) were generously provided by Dr Ko from Keck School of Medicine (Los Angeles, CA). PLC/PRF/5 hepatocellular carcinoma cells (p53 and K-Ras mutant) were purchased from ATCC (Manassas, VA). HuH-7 cells were cultured in MEM media (HyClone, Logan, UT) containing 10% fetal bovine serum (HyClone) and 1% penicillin-streptomycin (HyClone), whereas PLC/ PRF/5 were cultured in RPMI 1640 containing 1 mM pyruvate, 10% fetal bovine serum, and 1% penicillin-streptomycin. Cells were maintained at 378C in a humidified atmosphere with 5% CO2. Treatment of cells with triptolide and sorafenib. Triptolide (Calbiochem EMD Chemicals, Inc, Gibbstown NJ) was dissolved in dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO) and added to the cells at the indicated concentrations in serum-free medium (SFM). Sorafenib was a kind gift from Dr Edward Greeno at the University of Minnesota (Minneapolis, MN), and was prepared by dissolution in dimethyl sulfoxide. Cells treated with SFM alone served as controls. Determination of cell viability. Cells (HuH-7 seeded at 5 3 103/well, PLC seeded at 104/well) were cultured in 96-well plates and allowed to adhere for 4872 hours at 378C. Treatment with triptolide, sorafenib, or the combination of both at varying concentrations were carried out in SFM for 2472 hours. Cell viability was determined by the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) at respective time points. To summarize, 10 mL of the trazolium substrate was added to each well of the plate and allowed to incubate at 378C for 1 hour. Absorbance at 450 nm was measured, and the results were normalized to untreated cells at respective time points. Caspase 3/7 activity assay. Cells were cultured as stated previously into 96-well white opaque plates, as well as in a corresponding optically clear 96-well plate. Cells were allowed to adhere for 4872 hours at 378C. Treatments were carried
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out at varying concentrations of triptolide, sorafenib, or the combination of both for 24 or 48 hours. At the end of the incubation time, 100 mL of the appropriate Caspase-Glo reagent 3/7(Caspase Glo 3/7; Promega, Madison, WI) was added to each well containing 100 mL of blank, negative control or treated cells in serum-free medium. Plates were incubated in the dark for 1 hour at room temperature. The luminescence was then read. The corresponding 96-well clear plate was used to measure the number of viable cells with the CCK8 reagent, and caspase activity was normalized to these values. Nuclear factor kappa-B (NF-kB) reporter assay. Cells (HuH-7 or PLC/PRF/5) were cultured at 8 3 104/well into a 24-well clear plate and allowed to adhere for 24 hours. NF-kB Dual-Luciferase reporter assay system (QIAGEN, Valencia, CA) was used. To summarize, Attractene (QIAGEN) was used to form DNA plasmid complexes and added to the wells and allowed incubated at 378C for 24 hours. Treatment with triptolide, sorafenib, or a combination of both was then carried out. Cells were lysed using 1X passive lysis buffer (Dual-Glo kit; Promega) 68 hours posttreatment, and plates were then frozen at 808C for at least 1 hour. Luciferase activity (luminescence) of both inducible firefly and constitutive renilla constructs were then measured using the Dual-Glo kit (Promega). Heterotopic HCC animal model. Five athymic nude mice (nu/nu; NCI) were injected with 5 3 106 HuH-7 cells subcutaneously in the right flank in 100-mL aliquots mixed with Matrigel (BD Biosciences, San Jose, CA) in 1:1 ratio. Tumors were allowed to grow for 6 weeks, and then explanted, divided, and implanted into 40 athymic nude mice. This process ensured adequate and equal initial tumor size within the experimental groups. Animals were then randomized into four treatment groups (n = 10 in each group); (1) saline, (2) sorafenib 10 mg/kg, (3) Minnelide 0.21 mg/kg, and (4) combination (sorafenib 10 mg/kg and Minnelide 0.21 mg/kg). Sorafenib was dissolved in Cremophor (Sigma-Aldrich) and 100% ethanol in 1:1 ratio to make a 4X stock, which was discarded after 4 days. Stock solution was diluted in water before administration orally via disposable gavage needles in 200-mL aliquots daily. Minnelide was administered intraperitoneally in 200-mL aliquots. Treatment began at day 10 postimplant, and tumor dimensions and animal weight were measured weekly. Tumor volume was assessed using the following formula; (length 3 width 3 width 3 0.52).
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Fig 1. HuH-7 cell viability and caspase 3 and 7 activation assays. Triptolide (A) and sorafenib (B) decrease HuH-7 cell viability in a time- and dose-dependent fashion. The combination of triptolide 50 nM with sorafenib 1.25 mM was superior to single drug therapy in reducing cell viability at 72 hours (C, combination = 8 ± 1%, T50 = 20 ± 2%, and S1.25 = 26 ± 3%, zP < .01), and inducing caspase 3 and 7 activation at 48 hours (D, combination = 15-fold, T50 = 6.7-fold, and S1.25 = 2-fold increase over control, zP < .01). (*P < .05 for treatment group(s) compared to control.) Data are mean ± SEM.
Transcription factor enzyme-linked immunosorbent assay (ELISA) for NF-kB p50 subunit. p50 transcription factor binding using the p50 NF-kB ELISA kit was used to assess the levels of p50 subunit in tumor lysates (Thermo, Waltham, MA). In summary, 50 mL of working solution (ultrapure water, 5X NF-kB binding buffer, 20X Poly dl.dC) were added to the p50-coated ELISA strips (coated with consensus sequence that binds only the active form of p50) and incubated for 1 hour with mild agitation. Then, 100 mL of primary antibody (NF-kB p50) was added after washing three times and incubated for another 1 hour without agitation. 100 mL of the horseradish peroxidaseconjugated secondary antibody was added after washing for three times and incubated without agitation for an additional hour. Wells were then washed for 3 times, 100 mL of chemiluminescent substrate was added, and chemiluminescence was measured. Statistical analysis. Continuous variables are represented as mean ± standard error of the mean (SEM). Unpaired two-sample Student’s t test was used to discern differences between the
treatment groups. A P-value of less than 0.05 was considered statistically significant. StatPlus:Mac software (v 5.8.3.8) was used to perform the statistical analyses. RESULTS Combination of sorafenib and triptolide is superior to either drug treatment in inducing HCC cell death in vitro. There was a time- and dose-dependent increase in cell death after drug treatments. Compared with untreated cells at their respective time points, 50 nM concentration of triptolide decreased HuH-7 cell viability to 71 ± 3%, 45 ± 3%, and 20 ± 2% at 24, 48, and 72 hours, respectively (Fig 1, A). Further increasing the dose to 100 nM did not decrease cell viability substantially more; however, treatment with 1.25 mM sorafenib decreased cell viability to 49 ± 3%, 38 ± 3%, and 26 ± 3% at 24, 48, and 72 hours, respectively (Fig 1, B). When both drug treatments were combined, the resulting decrease in cell viability was superior at all three time points; 33 ± 3%, 18 ± 2%, and 8 ± 1% at 24, 48, and 72 hours, respectively (Fig 1, C). At 72 hours, all
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Fig 2. PLC/PRF/5 cell viability and caspase 3 and 7 activation assays. Triptolide (A) and sorafenib (B) decrease PLC/ PRF/5 cell viability in a time- and dose-dependent fashion. Combination of triptolide 50 nM with sorafenib 2.5 mM decreasing cell viability at 72 hours (C, combination = 31 ± 11%, T50 = 49 ± 9%, and S1.25 = 44 ± 13%), and inducing caspase 3 and 7 activation at 48 hours (D, combination = 12.6-fold, T50 = 4.4-fold, and S1.25 = 2.5-fold increase over control, ***P < .05). (*P < .01 for treatment group(s) compared to control, yP < .05 for T100/T200 compared withT50, zP < .05 for S2.5/S5 compared with S1.25, **P < .05 for treatment group(s) compared with control, ***P < .05 for combination compared with single treatment).
single drug treatments were superior to control (P < .05), whereas combination treatment was superior in inducing cell death to either drug alone (P < .01). For the PLC/PRF/5 cells, triptolide at 50 nM concentration decreased cell viability to 71 ± 2%, 53 ± 6%, and 49 ± 9% at 24, 48, and 72 hours, respectively (Fig 2, A). Increasing the dose to 100 nM decreased cell viability further to 51 ± 5%, 33 ± 7%, and 22 ± 8% (Fig 2, A). Treatment with 1.25 mM sorafenib had a negligible effect on cell viability, whereas increasing the dose to 2.5 mM reduced cell viability further to 79 ± 4%, 60 ± 10%, and 43 ± 13% at 24, 48, and 72 hours, respectively (Fig 2, B). Of note, the PLC/PFR/5 cells were more resistant to sorafenib compared with their HuH-7 counterparts (Figs 1, B and 2, B). When combined triptolide 50 nM and sorafenib 2.5 mM, the resulting decrease in cell viability was 56 ± 4%, 41 ± 10%, and 31 ± 13% at 24, 48, and 72 hours, respectively (Fig 1, C). At 72 hours, all single drug treatments were superior
to control (P < .05), whereas combination treatment was superior in inducing cell death to either drug alone at 24 hours (P < .05). Triptolide and sorafenib combination mediates apoptotic cell death in HCC cells. In HuH-7 cells, triptolide (50 nM) induced caspase 3 and 7 activity to 1.6- and 6.7-fold over control untreated cells, whereas sorafenib (1.25 mM, which corresponds to less than 10% of therapeutic dose in human patients) resulted in an increase of 1.6- and 2fold over control, for 24 and 48 hours, respectively. When both triptolide and sorafenib treatments were combined, caspase 3 and 7 increased to 3.7and 15-fold at 24 and 48 hours of treatment, respectively (Fig 1, D). At 48 hours, single drug treatments were superior to control (P < .05), whereas combination treatment was superior to either drug alone (P < .01). In PLC/PFR/5 cells, triptolide (50 nM) induced caspase 3 and 7 activity to 5.8- and 4.4-fold over control untreated cells, whereas sorafenib (2.5 mM) resulted an increase of 2.1- and 2.5-fold over
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Fig 3. NF-kB reporter assay. Triptolide decreased NF-kB activity in triptolide 50 nM and in combination treatments to 64 ± 6% and 66 ± 10%, respectively, in HuH7 cells (A) and 67 ± 7% and 54 ± 3% in PLC/PFR/5 cells (B). (*P < .05).
control, for 24 and 48 hours, respectively. In the combination treatment, caspase 3 and 7 increased to 9.7- and 12.6-fold at 24 and 48 hours, respectively (Fig 2, D). At 48 hours, single drug treatments were superior to control (P < .01), whereas combination treatment was superior to either drug alone (P < .05). Triptolide inhibits NF-kB activity in HCC cells in vitro. Triptolide decreased NF-kB binding to target element promoters as assessed by the NF-kB Dual-Luciferase reporter assay system. Triptolide alone at 50 nM or in combination with sorafenib (1.25 mM for HuH-7, 2.5 mM for PLC/PRF/5) produced a similar decrease in NF-kB activity at 64 ± 6% and 66 ± 10% compared with untreated control in HuH-7 cells, and 67 ± 7% and 54 ± 3% in PLC/PFR/5 cells after 68 hours of treatment (Fig 3, A and B). Treatment with sorafenib had no effect on NF-kB activity in either cell line. Combination of Minnelide and sorafenib is superior in inhibiting tumor progression in vivo. After implanting athymic nude mice with HuH7derived tumors at the right flank, tumor progression was assessed weekly in the four treatment groups (vehicle saline control orally [PO] daily,
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Fig 4. Tumor progression. At day 14 of treatment, saline group tumor volumes were 9.2-fold increased compared with 4.6-, 3.2-, and 1.5-fold for the sorafenib, Minnelide, and combination groups. Three mice from the saline group tumors were selected randomly and allowed to grow for three more days. Those tumors grow to 24fold their initial tumor size. The remaining seven mice were crossed over to receive combination treatment (Fig 7). At the conclusion of the experiment, combination group tumors were lower (4.3 ± 1.2-fold) than their sorafenib (9.4 ± 1.5-fold, P < .05), and Minnelide counterparts (13.4 ± 4.3-fold, P = .08). (*P < .001 for treatment groups against control, yP < .05 for combination treatment compared with sorafenib.)
sorafenib 10 mg/kg PO daily, Minnelide 0.21 mg/ kg intraperitoneally daily, and the combination of both drugs, n = 10 per group). The dose of sorafenib administered corresponds to in vitro concentrations of 1.4 mM, which is less than 10% of therapeutic dose in human patients. On day 14 of treatment, there was a divergence in efficacy of different treatments compared with saline control. Tumor volume increased rapidly in the control mice to reach 9.2-fold of the initial volume, whereas other treatment groups showed a much slower progression rate at 4.6-, 3.2-, and 1.5-fold, which corresponds to tumor growth inhibition rates of 59%, 84%, and 93% for the sorafenib, Minnelide, and combination treatments at 14 days (Fig 4). At the end of the experiment (6 weeks of treatment), all treatment groups showed a decrease in tumor compared with control mice (P < .001). On day 14 of treatment, because of a rapid increase in tumor volume of the control group, animals from this group (n = 7) were crossed over to combination treatment (Fig 7). After 6 weeks of treatment, sorafenib- and Minnelide-treated animals had similar-sized tumors, which were not statistically different (9.4 ± 1.5- vs 13.4 ± 2.6-fold initial volume), but tumor volumes of animals treated with a combination of sorafenib and Minnelide were less than the
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animals in the sorafenib group (4.3 ± 1.2 vs 9.4 ± 1.5 fold-initial volume, P < .05), but not significantly in the Minnelide group (4.3 ± 1.2 vs 13.4 ± 4.3 fold-initial volume, P = .08). On day 14 after start of treatment, three mice were sacrificed randomly from all treatment groups and tumors were assessed for tumor mass, volume, and NF-kB binding activity. Tumor volumes were reduced in all treatment groups compared with control mice (P < .01); the decrease was 76 ± 8%, 76 ± 3%, and 93 ± 4% for the sorafenib, Minnelide, and combination treatments, respectively (Fig 5, A). Combination group tumor volumes were less compared with Minnelide (P < .05) but not sorafenib (P = .1). Minnelide and sorafenib groups were not different (P = 1). Similarly, tumor mass was smaller in the treatment groups compared with control mice at 5.2 ± 0.4, 1.3 ± 0.4, 1.1 ± 0.2, and 0.4 ± 0.2 g for the control, sorafenib, Minnelide, and combination groups, respectively (P < .01; Fig 5, B). The difference in tumor mass did not differ between the combination and individual drug treatment groups (sorafenib, P = .1; Minnelide, P = .07). Nonetheless, this result is consistent with the pattern of enhanced tumor reduction in the combination treated mice compared with single drug treatments. We then examined the amount of dissociated (active) p50 in total tumor lysates by ELISA. The combination treatment decreased binding activity to 32 ± 20% (P = .06; Fig 5, C). Combination treatment with Minnelide and sorafenib inhibit tumor proliferation in vivo. Tumors from animals treated with a combination of sorafenib and Minnelide showed extensive destruction of tissue architecture and scar formation on hematoxylin and eosin staining, along with paucity of proliferating cells on Ki-67 staining (Fig 6). Treatment with either sorafenib or Minnelide alone showed some degree of cell death as evidenced by islands of cellular debris, but overall tissue architecture was not disturbed. Cellular proliferation was not different in animals from the control group compared with those in the single treatment groups, whereas combination treated tumors showed less Ki-67 staining (P < .05). Combination treatment with Minnelide and sorafenib prevents further growth of large, established tumors. After implanting athymic nude mice with HuH-7formed tumors in the right flank, tumor progression was assessed weekly in the two treatment groups (vehicle saline control PO daily and combination treatment with sorafenib 10 mg/kg PO + Minnelide 0.21 mg/kg intraperitoneally daily, n = 10 per group). Because
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Fig 5. Characteristics of tumors after 2 weeks of treatment (n = 3). Percent tumor decrease (A) was 76 ± 8%, 76 ± 3%, and 93 ± 4%, for the sorafenib, Minnelide, and combination groups, respectively. Corresponding tumor masses (B) were 5.2 ± 0.4, 1.3 ± 0.4, 1.1 ± 0.2, 0.4 ± 0.2 g, for the control, sorafenib, Minnelide, and combination groups, respectively (P < .05). NF-kB p50 transcription factor binding activity (C) assay values was decreased only in the combination treatment to 32 ± 20%, (yP = .06). (*P < .01 compared with the control group, yP = .06 for combination compared with all treatment groups. (Percent decrease = [average explanted treatment tumor volume/average explanted saline tumor volume] 3 100%).
rapid tumor growth in the control group, seven animals were crossed over to the combination arm of treatment (crossover group) on day 14 after experiment initiation. The remaining three mice were allowed to grow tumors for 3 more days, at which
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Fig 7. Effect of combination treatment on tumor growth after a 2-week growth lag phase. After crossing over to combination treatment, tumor volumes remained constant at 8-fold initial tumor volume for the remaining 4 weeks of treatment (A). Tumor mass was similar at the end of the experiment for the combination and crossover groups; 1.3 ± 0.5 and 1.2 ± 0.3 g, respectively (P = .9) compared with 5.2 ± 0.4 g in the control saline group (B). (* and yP < .01 compared against control group.)
Fig 6. Hematoxylin and eosin stain micrographs (A), Ki67 stain micrographs (B), Ki-67 intensity (C). (a) saline control, (b) sorafenib, (c) Minnelide, (d) combination. Both sorafenib and Minnelide alone showed some degree of cell death and decrease in Ki-67 staining; however, combination-treated tumors showed extensive destruction of tissue architecture and less Ki-67 staining (P < .05). *P < .05 for combination treatment compared with all other groups.
volume for the remaining 4 weeks of treatment after starting the combination therapy. Meanwhile, during the same time period, the tumor volumes in the combination treatment arm increased from 1.5- to 4.3-fold of initial tumor. Tumor mass was similar at the end of the experiment for the combination and crossover groups; 1.3 ± 0.5 and 1.2 ± 0.3 g, respectively (P = .9) compared with 5.2 ± 0.4 g in the control saline group (Fig 7, B). Both tumor volumes and mass were less in the combination group compared to saline controls (P < .01).
point they were sacrificed because tumor volume reached the limit permitted by the animal care committee at the University of Minnesota (Fig 7, A). Interestingly, the crossover group tumor volumes remained constant at 8-fold initial tumor
DISCUSSION HCC remains one of the deadliest solid tumors with no effective therapies.1,2,15 In the United States, there were an estimated 30,640 new cases of HCC resulting in 21,670 deaths in 2013.16
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Five-year survival remains poor at 28% for localized disease, 10% for regional disease, 3% for metastatic disease, and 15% for all stages combined.4,17 Viral hepatitis is the major risk factor leading to the development of HCC. Chronic hepatitis C infections account for 60% of HCC in Europe and North America, hepatitis B infections account for 70% of HCC in Asia and Africa, whereas alcoholic liver cirrhosis and other risk factors account for the remaining etiologies of HCC.1,3 No single genetic alteration is responsible for the emergence of HCC; the accumulation of epigenetic (APC/Wnt signaling), intracellular signaling (Mitogen-Activated Protein Kinases: RAF/MEK/ERK and phosphoinositide 3-kinase/ Akt/mTOR), and receptor/growth factor (EGFR/ EGF, HGF/c-MET, IGFR/IGF, VEGFR/angiopoietin) abnormalities combine in a multistep fashion with acute hepatitis, leading up to chronic hepatitis, cirrhosis, and nodular regeneration, and, eventually, hepatocellular carcinoma.18-20 Drug discovery for the extremely vascular HCC cancer has been guided by the concept of inhibiting angiogenesis.8 Sorafenib, a multikinase inhibitor, inhibits the RAF/MEK/ERK signaling pathway, as well as multiple tyrosine kinase receptors (VEGFR-2, VEGFR-3, platelet-derived growth factor receptor, Flt-3, and c-KIT).9,14,21,22 In 2008, sorafenib was approved by the US Food and Drug Administration as a targeted therapy for metastatic HCC treatment after the SHARP trial.5 In this phase 3 clinical trial, sorafenib increased median survival modestly from 7.9 to 10.7 months and time to radiographic progression from 2.8 to 5.5 months.5 Unfortunately, sorafenib has a number of disabling side effects at full dose that prevents patients from continuing therapy,22 which has led multiple investigators to attempt combining sorafenib with other agents to minimize the dose of sorafenib, thereby ameliorating its side effect profile as well as potentially target other pathway abnormalities.7,22-26 Triptolide and its prodrug Minnelide11,13 have an antineoplastic effect on multiple tumor types, including cholangiocarcinoma,27 pancreatic adenocarcinoma,11-13,28,29 osteosarcoma,30 lung adenocarcinoma,31 and neuroblastoma.32,33 Our group has also found that triptolide inhibits the phosphoinositide 3-kinase/Akt/ mTOR pathway,28 induces apoptosis,28 and decreases levels of heat shock proteins11,12,29 in pancreatic adenocarcinoma cells. Triptolide, the parent drug, is not water-soluble; however, Minnelide is water-soluble and safe for use in animals.11,13
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We hypothesized that combining sorafenib and triptolide (Minnelide for in vivo experiments) would maximize therapeutic effect and limit toxicity of either drug treatment alone. We showed here that a combination of these drugs at doses that are ineffective when given alone, is superior to either drug treatment in inducing cell death and committing HCC cells to apoptosis in vitro. A distinct advantage of using a combinational therapy is decreasing the dose of sorafenib, where we used 1.25–2.5 mM in our in vitro assays, which corresponds to 1025% of the therapeutic plasma level of an patient with HCC receiving sorafenib at a therapeutic dose of 400 mg twice daily orally.26 At the cellular level, Triptoldie inhibited the prosurvival transcription factor NF-kB in tumor cells. Many investigators suggest that NF-kB activation plays a pivotal role in the development of numerous malignancies by enhancing the local tumoral milieu supply of inflammatory mediators and maintaining chronic inflammatory status.34,35 NF-kB signaling is essential for many cellular functions, however, when constitutively activated in tumor cells, NF-kB signaling enhances the antiapoptotic protein levels as well as proangiogenic pathways.36,37 Also, this change in local tumor environment is believed to transform host innate immune components, such as macrophages, to defend and protect the tumors by evading cellular immune surveillance.34,35 We found that triptolide, but not sorafenib, inhibited basal NF-kB activity, and this effect carried forward into the combination treatment in vitro. Our in vivo data projects an even more compelling story that advocates the use of combinational therapy with sorafenib and Minnelide. We used a low dose of sorafenib (10 mg/kg, which is equivalent to 1.4 mM in vitro concentration26) and show that it induces a modest decrease in tumor volume and proliferation that validates the data published by the developers of sorafenib in 2006.9 More interestingly, we showed that a relatively low dose of Minnelide (0.21 mg/kg) produced the similar decrease in tumor volume and growth. When both drugs were combined, the results were well beyond the additive effect of either drug influence alone, which suggest a synergistic effect. This effect is observed especially in the inhibition of NF-kB binding in tumor xenograft samples. Although neither drug alone decreased p50 transcription factor binding in vivo, the use of combination therapy decreased the binding by 68%. We hypothesize that the use of two drugs that act on different pathways enhances antitumoral because they act in a complementary fashion
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Fig 8. Hypothesis. Sorafenib inhibits the RAF/MEK/ERK signaling pathway, whereas triptolide (Minnelide) inhibits the Akt/mTOR pathway and basal NF-kB activity/activation. Combined together, the two drugs act on different pathways to enhance antitumoral effects.
(Fig 8). Although sorafenib inhibits the RAF/MEK/ ERK signaling pathway, triptolide (Minnelide) inhibited NF-kB activation in a complementary fashion. Triptolide also decreased the mRNA levels of multiple tyrosine kinase receptors in vitro (Supplementary Fig 1; online version only). Most interestingly, we were able to show that combinational therapy is effective in limiting tumor progression over the period of 4-week treatment. It’s difficult to discern why combination treated tumors continued to grow, albeit at a very slow rate, whereas the crossover treated tumors had plateaued during period of treatment (Fig 7). This observation was especially true in a subcutaneous heterotopic tumor model. One possibility is that crossover-treated tumors reached a critical volume at which the treatment-induced decrease in vessel density did not allow further tumor growth, while the combination treated tumors volumes were initially less. This finding could also be a cell-specific phenomenon. To answer this question, we also carried out a similar experiment wherein 36 mice were implanted with PLC/PRF/5-derived tumors and divided into four groups (n = 8); saline, sorafenib (10 mg/kg), Minnelide (0.21 mg/kg), and combination. Combination treatment induced tumor regression to 30% of original tumor size, whereas Minnelide and sorafenib slowed tumor progression to 1.3 ± 0.5- and 3.2 ± 1.1-fold compared with 5.9 ± 3-fold growth in the saline group. When crossed over to combination treatment, saline group tumors regressed to 3 ± 1.4-fold over a 24-day of treatment from 5.9 ± 3 fold; an overall
49% decrease in tumor volumes (Supplementary Fig 2; online version only). Our crossover experiment suggests that the combination of sorafenib and Minnelide not only prevented tumor progression but also caused tumor regression, which is most relevant because it mimics locoregionally metastatic disease. SUPPLEMENTARY DATA Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.surg.2014. 04.055. REFERENCES 1. Forner A, Llovet JM, Bruix J. Hepatocellular carcinoma. Lancet 2012;379:1245-55. 2. Finn RS. Current and future treatment strategies for patients with advanced hepatocellular carcinoma: role of mTOR inhibition. Liver Cancer 2012;1:247-56. 3. El-Serag HB, Davila JA, Petersen NJ, McGlynn KA. The continuing increase in the incidence of hepatocellular carcinoma in the United States: an update. Ann Intern Med 2003;139:817-23. 4. American Cancer Society. Liver Cancer. Chicago, IL: American Cancer Society; 2012. 5. Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, et al. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 2008;359:378-90. 6. Salem R, Hunter RD. Yttrium-90 microspheres for the treatment of hepatocellular carcinoma: a review. Int J Radiat Oncol Biol Phys 2006;66:S83-8. 7. Gedaly R, Angulo P, Hundley J, Daily MF, Chen C, Evers BM. PKI-587 and sorafenib targeting PI3K/AKT/mTOR and Ras/Raf/MAPK pathways synergistically inhibit HCC cell proliferation. J Surg Res 2012;176:542-8. 8. Tanaka S, Arii S. Molecular targeted therapies in hepatocellular carcinoma. Semin Oncol 2012;39:486-92.
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9. Liu L, Cao Y, Chen C, Zhang X, McNabola A, Wilkie D, et al. Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5. Cancer Res 2006;66:11851-8. 10. Shi YH, Ding ZB, Zhou J, Hui B, Shi GM, Ke AW, et al. Targeting autophagy enhances sorafenib lethality for hepatocellular carcinoma via ER stress-related apoptosis. Autophagy 2011;7:1159-72. 11. Phillips PA, Dudeja V, McCarroll JA, Borja-Cacho D, Dawra RK, Grizzle WE, et al. Triptolide induces pancreatic cancer cell death via inhibition of heat shock protein 70. Cancer Res 2007;67:9407-16. 12. Aghdassi A, Phillips P, Dudeja V, Dhaulakhandi D, Sharif R, Dawra R, et al. Heat shock protein 70 increases tumorigenicity and inhibits apoptosis in pancreatic adenocarcinoma. Cancer Res 2007;67:616-25. 13. Chugh R, Sangwan V, Patil SP, Dudeja V, Dawra RK, Banerjee S, et al. A preclinical evaluation of minnelide as a therapeutic agent against pancreatic cancer. Sci Transl Med 2012;4:156ra39. 14. Nagai T, Arao T, Furuta K, Sakai K, Kudo K, Kaneda H, et al. Sorafenib inhibits the hepatocyte growth factor-mediated epithelial mesenchymal transition in hepatocellular carcinoma. Mol Cancer Ther 2011;10:169-77. 15. World Health Organization. Cancer. Available from http:// www.who.int/mediacentre/factsheets/fs297/en/. 16. American Cancer Society. Cancer figures & facts 2013. Chicago: American Cancer Society; 2013. 17. National Cancer Institute. Surveillence, Epidemiology, and End Results Program. Available from: http://seer.cancer. gov/statfacts/html/livibd.html. 18. Wong CM, Ng IO. Molecular pathogenesis of hepatocellular carcinoma. Liver Int 2008;28:160-74. 19. Llovet JM, Bruix J. Molecular targeted therapies in hepatocellular carcinoma. Hepatology 2008;48:1312-27. 20. Huynh H. Molecularly targeted therapy in hepatocellular carcinoma. Biochem Pharmacol 2010;80:550-60. 21. Wilhelm SM, Carter C, Tang L, Wilkie D, McNabola A, Rong H, et al. BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer Res 2004;64:7099-109. 22. Xie B, Wang DH, Spechler SJ. Sorafenib for treatment of hepatocellular carcinoma: a systematic review. Dig Dis Sci 2012;57:1122-9. 23. Chen KF, Tai WT, Liu TH, Huang HP, Lin YC, Shiau CW, et al. Sorafenib overcomes TRAIL resistance of hepatocellular carcinoma cells through the inhibition of STAT3. Clin Cancer Res 2010;16:5189-99. 24. Gedaly R, Angulo P, Hundley J, Daily MF, Chen C, Koch A, et al. PI-103 and sorafenib inhibit hepatocellular carcinoma cell proliferation by blocking Ras/Raf/MAPK and
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PI3K/AKT/mTOR pathways. Anticancer Res 2010;30: 4951-8. Hikita H, Takehara T, Shimizu S, Kodama T, Shigekawa M, Iwase K, et al. The Bcl-xL inhibitor, ABT-737, efficiently induces apoptosis and suppresses growth of hepatoma cells in combination with sorafenib. Hepatology 2010;52: 1310-21. Hu S, Niu H, Inaba H, Orwick S, Rose C, Panetta JC, et al. Activity of the multikinase inhibitor sorafenib in combination with cytarabine in acute myeloid leukemia. J Natl Cancer Inst 2011;103:893-905. Clawson KA, Borja-Cacho D, Antonoff MB, Saluja AK, Vickers SM. Triptolide and TRAIL combination enhances apoptosis in cholangiocarcinoma. J Surg Res 2010;163: 244-9. Mujumdar N, Mackenzie TN, Dudeja V, Chugh R, Antonoff MB, Borja-Cacho D, et al. Triptolide induces cell death in pancreatic cancer cells by apoptotic and autophagic pathways. Gastroenterology 2010;139:598-608. Dudeja V, Chugh RK, Sangwan V, Skube SJ, Mujumdar NR, Antonoff MB, et al. Prosurvival role of heat shock factor 1 in the pathogenesis of pancreatobiliary tumors. Am J Physiol Gastrointest Liver Physiol 2011; 300:G948-55. Banerjee S, Thayanithy V, Sangwan V, Mackenzie TN, Saluja AK, Subramanian S. Minnelide reduces tumor burden in preclinical models of osteosarcoma. Cancer Lett 2013;335: 412-20. Rousalova I, Banerjee S, Sangwan V, Evenson K, McCauley JA, Kratzke R, et al. Minnelide: a novel therapeutic that promotes apoptosis in non-small cell lung carcinoma in vivo. PLoS ONE 2013;8:e77411. Antonoff MB, Chugh R, Borja-Cacho D, Dudeja V, Clawson KA, Skube SJ, et al. Triptolide therapy for neuroblastoma decreases cell viability in vitro and inhibits tumor growth in vivo. Surgery 2009;146:282-90. Krosch TC, Sangwan V, Banerjee S, Mujumdar N, Dudeja V, Saluja AK, et al. Triptolide-mediated cell death in neuroblastoma occurs by both apoptosis and autophagy pathways and results in inhibition of nuclear factor-kappa B activity. Am J Surg 2013;205:387-96. Beyaert R, Beaugerie L, Van Assche G, Brochez L, Renauld JC, Viguier M, et al. Cancer risk in immune-mediated inflammatory diseases (IMID). Mol Cancer 2013;12:98. Ben-Neriah Y, Karin M. Inflammation meets cancer, with NF-kappaB as the matchmaker. Nat Immunol 2011;12: 715-23. Chaturvedi MM, Sung B, Yadav VR, Kannappan R, Aggarwal BB. NF-kappaB addiction and its role in cancer: ‘one size does not fit all’. Oncogene 2011;30:1615-30. Hoesel B, Schmid JA. The complexity of NF-kappaB signaling in inflammation and cancer. Mol Cancer 2013; 12:86.
Introduction. Sorafenib is the only drug approved by the Food and Drug Administration for metastatic hepatocellular carcinoma (HCC). Triptolide, a diterpene triepoxide, exhibits antineoplastic properties in multiple tumor cell types. In this study, we examined the effects of these agents and their combination on HCC in vitro and in vivo models. Methods. HuH-7 and PLC/PRF/5 cells were treated with triptolide (50 nM), sorafenib (1.25 or 2.5 mM), or a combination of both. Cell viability assay (CCK-8), caspase 3&7 activation, and nuclear factor kB assays were performed. For in vivo studies, 40 mice were implanted with subcutaneous HuH7 tumors and divided into four treatment groups (n = 10); saline control, sorafenib 10 mg/kg PO daily (S), Minnelide (a prodrug of triptolide) 0.21 mg/kg intraperitoneally7 daily (M), and combination of both (C). Tumor volumes were assessed weekly. Results. The combination of triptolide and sorafenib was superior to either drug alone in inducing apoptosis and decreasing viability, whereas triptolide alone was sufficient to decrease nuclear factor kB activity. After 2 weeks of treatment, tumor growth inhibition rates were S = 59%, M = 84%, and C = 93%, whereas tumor volumes in control animals increased by 9-fold. When crossed over to combination treatment, control mice tumor growth volumes plateaued over the following 4 weeks. Conclusion. The combination of sorafenib and triptolide is superior to single drug treatment in increasing cell death and apoptosis in vitro. Combining sorafenib with Minnelide inhibited tumor growth with greater efficacy than single-agent treatments. Importantly, in vivo combination treatment allowed for using a lesser dose of sorafenib (10 mg/kg), which is less than 10% of currently prescribed dose for HCC patients. Therefore, combination treatment could have translational potential in the management of HCC. (Surgery 2014;156:270-9.) From the Division of General Surgery,a Division of Basic and Translational Research,b and Division of Surgical Oncology, Department of Surgery, d University of Minnesota, Minneapolis, MN; and School of Medicine,c University of Alabama, Birmingham, AL
HEPATOCELLULAR CARCINOMA (HCC) is currently the sixth most-common cancer and is the thirdleading cause of cancer-related deaths worldwide.1,2 Chronic liver disease secondary to viral hepatitis is responsible for the majority of HCC Dr Saluja has a significant interest in and is the Chief Scientific Officer and a consultant for Minneamrita, a company that may commercially benefit from the results of this research. This relationship has been reviewed and managed by the University of Minnesota in accordance with its conflict of interest policies. Presented at the 9th Annual Academic Surgical Congress in San Diego, CA, February 4–6, 2014. Accepted for publication April 28, 2014. Reprint requests: Eric H. Jensen, MD, Division of Surgical Oncology, University of Minnesota Medical School, Minneapolis, MN 55455. E-mail: [email protected] 0039-6060/$ - see front matter Ó 2014 Mosby, Inc. All rights reserved. http://dx.doi.org/10.1016/j.surg.2014.04.055
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cases.1 In the United States, the incidence of HCC is related closely to hepatitis C infection and has been increasing during the past four decades.3 The prognosis of HCC is very poor with a 5-year survival for all stages at 15%, but with successful resection or liver transplantation, 5-year survival increases to 50% and 70%, respectively.4 Nonetheless, curative operative therapy is only offered to 1530% of all patients because of the advanced nature of the disease at the time of diagnosis.5,6 In 2007, the Sorafenib HCC Assessment Randomized Protocol Trial (SHARP) trial showed promising results with sorafenib, a multikinase inhibitor drug that increases median survival of patients with advanced HCC.5 Sorafenib targets a wide variety of growth factor and angiogenesis receptors, such as vascular endothelial growth factor receptor (VEGFR)2, VEGFR3, platelet-derived
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growth factor receptor, and c-KIT and affects multiple signal transduction pathways.7-10 The mechanism of action of sorafenib remains unclear. Minnelide is a water-soluble prodrug of triptolide, a diterpene triepoxide, derived from the Chinese herb Tripterygium wilfordii. Triptolide has been used in China to treat inflammatory conditions and cancers. Our group is currently investigating Minnelide in a phase 1 clinical trial for its antitumoral activity in gastrointestinal cancers.11-13 METHODS Cell culture. HuH-7 hepatocellular carcinoma cells (p53 mutant) were generously provided by Dr Ko from Keck School of Medicine (Los Angeles, CA). PLC/PRF/5 hepatocellular carcinoma cells (p53 and K-Ras mutant) were purchased from ATCC (Manassas, VA). HuH-7 cells were cultured in MEM media (HyClone, Logan, UT) containing 10% fetal bovine serum (HyClone) and 1% penicillin-streptomycin (HyClone), whereas PLC/ PRF/5 were cultured in RPMI 1640 containing 1 mM pyruvate, 10% fetal bovine serum, and 1% penicillin-streptomycin. Cells were maintained at 378C in a humidified atmosphere with 5% CO2. Treatment of cells with triptolide and sorafenib. Triptolide (Calbiochem EMD Chemicals, Inc, Gibbstown NJ) was dissolved in dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO) and added to the cells at the indicated concentrations in serum-free medium (SFM). Sorafenib was a kind gift from Dr Edward Greeno at the University of Minnesota (Minneapolis, MN), and was prepared by dissolution in dimethyl sulfoxide. Cells treated with SFM alone served as controls. Determination of cell viability. Cells (HuH-7 seeded at 5 3 103/well, PLC seeded at 104/well) were cultured in 96-well plates and allowed to adhere for 4872 hours at 378C. Treatment with triptolide, sorafenib, or the combination of both at varying concentrations were carried out in SFM for 2472 hours. Cell viability was determined by the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) at respective time points. To summarize, 10 mL of the trazolium substrate was added to each well of the plate and allowed to incubate at 378C for 1 hour. Absorbance at 450 nm was measured, and the results were normalized to untreated cells at respective time points. Caspase 3/7 activity assay. Cells were cultured as stated previously into 96-well white opaque plates, as well as in a corresponding optically clear 96-well plate. Cells were allowed to adhere for 4872 hours at 378C. Treatments were carried
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out at varying concentrations of triptolide, sorafenib, or the combination of both for 24 or 48 hours. At the end of the incubation time, 100 mL of the appropriate Caspase-Glo reagent 3/7(Caspase Glo 3/7; Promega, Madison, WI) was added to each well containing 100 mL of blank, negative control or treated cells in serum-free medium. Plates were incubated in the dark for 1 hour at room temperature. The luminescence was then read. The corresponding 96-well clear plate was used to measure the number of viable cells with the CCK8 reagent, and caspase activity was normalized to these values. Nuclear factor kappa-B (NF-kB) reporter assay. Cells (HuH-7 or PLC/PRF/5) were cultured at 8 3 104/well into a 24-well clear plate and allowed to adhere for 24 hours. NF-kB Dual-Luciferase reporter assay system (QIAGEN, Valencia, CA) was used. To summarize, Attractene (QIAGEN) was used to form DNA plasmid complexes and added to the wells and allowed incubated at 378C for 24 hours. Treatment with triptolide, sorafenib, or a combination of both was then carried out. Cells were lysed using 1X passive lysis buffer (Dual-Glo kit; Promega) 68 hours posttreatment, and plates were then frozen at 808C for at least 1 hour. Luciferase activity (luminescence) of both inducible firefly and constitutive renilla constructs were then measured using the Dual-Glo kit (Promega). Heterotopic HCC animal model. Five athymic nude mice (nu/nu; NCI) were injected with 5 3 106 HuH-7 cells subcutaneously in the right flank in 100-mL aliquots mixed with Matrigel (BD Biosciences, San Jose, CA) in 1:1 ratio. Tumors were allowed to grow for 6 weeks, and then explanted, divided, and implanted into 40 athymic nude mice. This process ensured adequate and equal initial tumor size within the experimental groups. Animals were then randomized into four treatment groups (n = 10 in each group); (1) saline, (2) sorafenib 10 mg/kg, (3) Minnelide 0.21 mg/kg, and (4) combination (sorafenib 10 mg/kg and Minnelide 0.21 mg/kg). Sorafenib was dissolved in Cremophor (Sigma-Aldrich) and 100% ethanol in 1:1 ratio to make a 4X stock, which was discarded after 4 days. Stock solution was diluted in water before administration orally via disposable gavage needles in 200-mL aliquots daily. Minnelide was administered intraperitoneally in 200-mL aliquots. Treatment began at day 10 postimplant, and tumor dimensions and animal weight were measured weekly. Tumor volume was assessed using the following formula; (length 3 width 3 width 3 0.52).
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Fig 1. HuH-7 cell viability and caspase 3 and 7 activation assays. Triptolide (A) and sorafenib (B) decrease HuH-7 cell viability in a time- and dose-dependent fashion. The combination of triptolide 50 nM with sorafenib 1.25 mM was superior to single drug therapy in reducing cell viability at 72 hours (C, combination = 8 ± 1%, T50 = 20 ± 2%, and S1.25 = 26 ± 3%, zP < .01), and inducing caspase 3 and 7 activation at 48 hours (D, combination = 15-fold, T50 = 6.7-fold, and S1.25 = 2-fold increase over control, zP < .01). (*P < .05 for treatment group(s) compared to control.) Data are mean ± SEM.
Transcription factor enzyme-linked immunosorbent assay (ELISA) for NF-kB p50 subunit. p50 transcription factor binding using the p50 NF-kB ELISA kit was used to assess the levels of p50 subunit in tumor lysates (Thermo, Waltham, MA). In summary, 50 mL of working solution (ultrapure water, 5X NF-kB binding buffer, 20X Poly dl.dC) were added to the p50-coated ELISA strips (coated with consensus sequence that binds only the active form of p50) and incubated for 1 hour with mild agitation. Then, 100 mL of primary antibody (NF-kB p50) was added after washing three times and incubated for another 1 hour without agitation. 100 mL of the horseradish peroxidaseconjugated secondary antibody was added after washing for three times and incubated without agitation for an additional hour. Wells were then washed for 3 times, 100 mL of chemiluminescent substrate was added, and chemiluminescence was measured. Statistical analysis. Continuous variables are represented as mean ± standard error of the mean (SEM). Unpaired two-sample Student’s t test was used to discern differences between the
treatment groups. A P-value of less than 0.05 was considered statistically significant. StatPlus:Mac software (v 5.8.3.8) was used to perform the statistical analyses. RESULTS Combination of sorafenib and triptolide is superior to either drug treatment in inducing HCC cell death in vitro. There was a time- and dose-dependent increase in cell death after drug treatments. Compared with untreated cells at their respective time points, 50 nM concentration of triptolide decreased HuH-7 cell viability to 71 ± 3%, 45 ± 3%, and 20 ± 2% at 24, 48, and 72 hours, respectively (Fig 1, A). Further increasing the dose to 100 nM did not decrease cell viability substantially more; however, treatment with 1.25 mM sorafenib decreased cell viability to 49 ± 3%, 38 ± 3%, and 26 ± 3% at 24, 48, and 72 hours, respectively (Fig 1, B). When both drug treatments were combined, the resulting decrease in cell viability was superior at all three time points; 33 ± 3%, 18 ± 2%, and 8 ± 1% at 24, 48, and 72 hours, respectively (Fig 1, C). At 72 hours, all
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Fig 2. PLC/PRF/5 cell viability and caspase 3 and 7 activation assays. Triptolide (A) and sorafenib (B) decrease PLC/ PRF/5 cell viability in a time- and dose-dependent fashion. Combination of triptolide 50 nM with sorafenib 2.5 mM decreasing cell viability at 72 hours (C, combination = 31 ± 11%, T50 = 49 ± 9%, and S1.25 = 44 ± 13%), and inducing caspase 3 and 7 activation at 48 hours (D, combination = 12.6-fold, T50 = 4.4-fold, and S1.25 = 2.5-fold increase over control, ***P < .05). (*P < .01 for treatment group(s) compared to control, yP < .05 for T100/T200 compared withT50, zP < .05 for S2.5/S5 compared with S1.25, **P < .05 for treatment group(s) compared with control, ***P < .05 for combination compared with single treatment).
single drug treatments were superior to control (P < .05), whereas combination treatment was superior in inducing cell death to either drug alone (P < .01). For the PLC/PRF/5 cells, triptolide at 50 nM concentration decreased cell viability to 71 ± 2%, 53 ± 6%, and 49 ± 9% at 24, 48, and 72 hours, respectively (Fig 2, A). Increasing the dose to 100 nM decreased cell viability further to 51 ± 5%, 33 ± 7%, and 22 ± 8% (Fig 2, A). Treatment with 1.25 mM sorafenib had a negligible effect on cell viability, whereas increasing the dose to 2.5 mM reduced cell viability further to 79 ± 4%, 60 ± 10%, and 43 ± 13% at 24, 48, and 72 hours, respectively (Fig 2, B). Of note, the PLC/PFR/5 cells were more resistant to sorafenib compared with their HuH-7 counterparts (Figs 1, B and 2, B). When combined triptolide 50 nM and sorafenib 2.5 mM, the resulting decrease in cell viability was 56 ± 4%, 41 ± 10%, and 31 ± 13% at 24, 48, and 72 hours, respectively (Fig 1, C). At 72 hours, all single drug treatments were superior
to control (P < .05), whereas combination treatment was superior in inducing cell death to either drug alone at 24 hours (P < .05). Triptolide and sorafenib combination mediates apoptotic cell death in HCC cells. In HuH-7 cells, triptolide (50 nM) induced caspase 3 and 7 activity to 1.6- and 6.7-fold over control untreated cells, whereas sorafenib (1.25 mM, which corresponds to less than 10% of therapeutic dose in human patients) resulted in an increase of 1.6- and 2fold over control, for 24 and 48 hours, respectively. When both triptolide and sorafenib treatments were combined, caspase 3 and 7 increased to 3.7and 15-fold at 24 and 48 hours of treatment, respectively (Fig 1, D). At 48 hours, single drug treatments were superior to control (P < .05), whereas combination treatment was superior to either drug alone (P < .01). In PLC/PFR/5 cells, triptolide (50 nM) induced caspase 3 and 7 activity to 5.8- and 4.4-fold over control untreated cells, whereas sorafenib (2.5 mM) resulted an increase of 2.1- and 2.5-fold over
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Fig 3. NF-kB reporter assay. Triptolide decreased NF-kB activity in triptolide 50 nM and in combination treatments to 64 ± 6% and 66 ± 10%, respectively, in HuH7 cells (A) and 67 ± 7% and 54 ± 3% in PLC/PFR/5 cells (B). (*P < .05).
control, for 24 and 48 hours, respectively. In the combination treatment, caspase 3 and 7 increased to 9.7- and 12.6-fold at 24 and 48 hours, respectively (Fig 2, D). At 48 hours, single drug treatments were superior to control (P < .01), whereas combination treatment was superior to either drug alone (P < .05). Triptolide inhibits NF-kB activity in HCC cells in vitro. Triptolide decreased NF-kB binding to target element promoters as assessed by the NF-kB Dual-Luciferase reporter assay system. Triptolide alone at 50 nM or in combination with sorafenib (1.25 mM for HuH-7, 2.5 mM for PLC/PRF/5) produced a similar decrease in NF-kB activity at 64 ± 6% and 66 ± 10% compared with untreated control in HuH-7 cells, and 67 ± 7% and 54 ± 3% in PLC/PFR/5 cells after 68 hours of treatment (Fig 3, A and B). Treatment with sorafenib had no effect on NF-kB activity in either cell line. Combination of Minnelide and sorafenib is superior in inhibiting tumor progression in vivo. After implanting athymic nude mice with HuH7derived tumors at the right flank, tumor progression was assessed weekly in the four treatment groups (vehicle saline control orally [PO] daily,
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Fig 4. Tumor progression. At day 14 of treatment, saline group tumor volumes were 9.2-fold increased compared with 4.6-, 3.2-, and 1.5-fold for the sorafenib, Minnelide, and combination groups. Three mice from the saline group tumors were selected randomly and allowed to grow for three more days. Those tumors grow to 24fold their initial tumor size. The remaining seven mice were crossed over to receive combination treatment (Fig 7). At the conclusion of the experiment, combination group tumors were lower (4.3 ± 1.2-fold) than their sorafenib (9.4 ± 1.5-fold, P < .05), and Minnelide counterparts (13.4 ± 4.3-fold, P = .08). (*P < .001 for treatment groups against control, yP < .05 for combination treatment compared with sorafenib.)
sorafenib 10 mg/kg PO daily, Minnelide 0.21 mg/ kg intraperitoneally daily, and the combination of both drugs, n = 10 per group). The dose of sorafenib administered corresponds to in vitro concentrations of 1.4 mM, which is less than 10% of therapeutic dose in human patients. On day 14 of treatment, there was a divergence in efficacy of different treatments compared with saline control. Tumor volume increased rapidly in the control mice to reach 9.2-fold of the initial volume, whereas other treatment groups showed a much slower progression rate at 4.6-, 3.2-, and 1.5-fold, which corresponds to tumor growth inhibition rates of 59%, 84%, and 93% for the sorafenib, Minnelide, and combination treatments at 14 days (Fig 4). At the end of the experiment (6 weeks of treatment), all treatment groups showed a decrease in tumor compared with control mice (P < .001). On day 14 of treatment, because of a rapid increase in tumor volume of the control group, animals from this group (n = 7) were crossed over to combination treatment (Fig 7). After 6 weeks of treatment, sorafenib- and Minnelide-treated animals had similar-sized tumors, which were not statistically different (9.4 ± 1.5- vs 13.4 ± 2.6-fold initial volume), but tumor volumes of animals treated with a combination of sorafenib and Minnelide were less than the
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animals in the sorafenib group (4.3 ± 1.2 vs 9.4 ± 1.5 fold-initial volume, P < .05), but not significantly in the Minnelide group (4.3 ± 1.2 vs 13.4 ± 4.3 fold-initial volume, P = .08). On day 14 after start of treatment, three mice were sacrificed randomly from all treatment groups and tumors were assessed for tumor mass, volume, and NF-kB binding activity. Tumor volumes were reduced in all treatment groups compared with control mice (P < .01); the decrease was 76 ± 8%, 76 ± 3%, and 93 ± 4% for the sorafenib, Minnelide, and combination treatments, respectively (Fig 5, A). Combination group tumor volumes were less compared with Minnelide (P < .05) but not sorafenib (P = .1). Minnelide and sorafenib groups were not different (P = 1). Similarly, tumor mass was smaller in the treatment groups compared with control mice at 5.2 ± 0.4, 1.3 ± 0.4, 1.1 ± 0.2, and 0.4 ± 0.2 g for the control, sorafenib, Minnelide, and combination groups, respectively (P < .01; Fig 5, B). The difference in tumor mass did not differ between the combination and individual drug treatment groups (sorafenib, P = .1; Minnelide, P = .07). Nonetheless, this result is consistent with the pattern of enhanced tumor reduction in the combination treated mice compared with single drug treatments. We then examined the amount of dissociated (active) p50 in total tumor lysates by ELISA. The combination treatment decreased binding activity to 32 ± 20% (P = .06; Fig 5, C). Combination treatment with Minnelide and sorafenib inhibit tumor proliferation in vivo. Tumors from animals treated with a combination of sorafenib and Minnelide showed extensive destruction of tissue architecture and scar formation on hematoxylin and eosin staining, along with paucity of proliferating cells on Ki-67 staining (Fig 6). Treatment with either sorafenib or Minnelide alone showed some degree of cell death as evidenced by islands of cellular debris, but overall tissue architecture was not disturbed. Cellular proliferation was not different in animals from the control group compared with those in the single treatment groups, whereas combination treated tumors showed less Ki-67 staining (P < .05). Combination treatment with Minnelide and sorafenib prevents further growth of large, established tumors. After implanting athymic nude mice with HuH-7formed tumors in the right flank, tumor progression was assessed weekly in the two treatment groups (vehicle saline control PO daily and combination treatment with sorafenib 10 mg/kg PO + Minnelide 0.21 mg/kg intraperitoneally daily, n = 10 per group). Because
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Fig 5. Characteristics of tumors after 2 weeks of treatment (n = 3). Percent tumor decrease (A) was 76 ± 8%, 76 ± 3%, and 93 ± 4%, for the sorafenib, Minnelide, and combination groups, respectively. Corresponding tumor masses (B) were 5.2 ± 0.4, 1.3 ± 0.4, 1.1 ± 0.2, 0.4 ± 0.2 g, for the control, sorafenib, Minnelide, and combination groups, respectively (P < .05). NF-kB p50 transcription factor binding activity (C) assay values was decreased only in the combination treatment to 32 ± 20%, (yP = .06). (*P < .01 compared with the control group, yP = .06 for combination compared with all treatment groups. (Percent decrease = [average explanted treatment tumor volume/average explanted saline tumor volume] 3 100%).
rapid tumor growth in the control group, seven animals were crossed over to the combination arm of treatment (crossover group) on day 14 after experiment initiation. The remaining three mice were allowed to grow tumors for 3 more days, at which
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Fig 7. Effect of combination treatment on tumor growth after a 2-week growth lag phase. After crossing over to combination treatment, tumor volumes remained constant at 8-fold initial tumor volume for the remaining 4 weeks of treatment (A). Tumor mass was similar at the end of the experiment for the combination and crossover groups; 1.3 ± 0.5 and 1.2 ± 0.3 g, respectively (P = .9) compared with 5.2 ± 0.4 g in the control saline group (B). (* and yP < .01 compared against control group.)
Fig 6. Hematoxylin and eosin stain micrographs (A), Ki67 stain micrographs (B), Ki-67 intensity (C). (a) saline control, (b) sorafenib, (c) Minnelide, (d) combination. Both sorafenib and Minnelide alone showed some degree of cell death and decrease in Ki-67 staining; however, combination-treated tumors showed extensive destruction of tissue architecture and less Ki-67 staining (P < .05). *P < .05 for combination treatment compared with all other groups.
volume for the remaining 4 weeks of treatment after starting the combination therapy. Meanwhile, during the same time period, the tumor volumes in the combination treatment arm increased from 1.5- to 4.3-fold of initial tumor. Tumor mass was similar at the end of the experiment for the combination and crossover groups; 1.3 ± 0.5 and 1.2 ± 0.3 g, respectively (P = .9) compared with 5.2 ± 0.4 g in the control saline group (Fig 7, B). Both tumor volumes and mass were less in the combination group compared to saline controls (P < .01).
point they were sacrificed because tumor volume reached the limit permitted by the animal care committee at the University of Minnesota (Fig 7, A). Interestingly, the crossover group tumor volumes remained constant at 8-fold initial tumor
DISCUSSION HCC remains one of the deadliest solid tumors with no effective therapies.1,2,15 In the United States, there were an estimated 30,640 new cases of HCC resulting in 21,670 deaths in 2013.16
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Five-year survival remains poor at 28% for localized disease, 10% for regional disease, 3% for metastatic disease, and 15% for all stages combined.4,17 Viral hepatitis is the major risk factor leading to the development of HCC. Chronic hepatitis C infections account for 60% of HCC in Europe and North America, hepatitis B infections account for 70% of HCC in Asia and Africa, whereas alcoholic liver cirrhosis and other risk factors account for the remaining etiologies of HCC.1,3 No single genetic alteration is responsible for the emergence of HCC; the accumulation of epigenetic (APC/Wnt signaling), intracellular signaling (Mitogen-Activated Protein Kinases: RAF/MEK/ERK and phosphoinositide 3-kinase/ Akt/mTOR), and receptor/growth factor (EGFR/ EGF, HGF/c-MET, IGFR/IGF, VEGFR/angiopoietin) abnormalities combine in a multistep fashion with acute hepatitis, leading up to chronic hepatitis, cirrhosis, and nodular regeneration, and, eventually, hepatocellular carcinoma.18-20 Drug discovery for the extremely vascular HCC cancer has been guided by the concept of inhibiting angiogenesis.8 Sorafenib, a multikinase inhibitor, inhibits the RAF/MEK/ERK signaling pathway, as well as multiple tyrosine kinase receptors (VEGFR-2, VEGFR-3, platelet-derived growth factor receptor, Flt-3, and c-KIT).9,14,21,22 In 2008, sorafenib was approved by the US Food and Drug Administration as a targeted therapy for metastatic HCC treatment after the SHARP trial.5 In this phase 3 clinical trial, sorafenib increased median survival modestly from 7.9 to 10.7 months and time to radiographic progression from 2.8 to 5.5 months.5 Unfortunately, sorafenib has a number of disabling side effects at full dose that prevents patients from continuing therapy,22 which has led multiple investigators to attempt combining sorafenib with other agents to minimize the dose of sorafenib, thereby ameliorating its side effect profile as well as potentially target other pathway abnormalities.7,22-26 Triptolide and its prodrug Minnelide11,13 have an antineoplastic effect on multiple tumor types, including cholangiocarcinoma,27 pancreatic adenocarcinoma,11-13,28,29 osteosarcoma,30 lung adenocarcinoma,31 and neuroblastoma.32,33 Our group has also found that triptolide inhibits the phosphoinositide 3-kinase/Akt/ mTOR pathway,28 induces apoptosis,28 and decreases levels of heat shock proteins11,12,29 in pancreatic adenocarcinoma cells. Triptolide, the parent drug, is not water-soluble; however, Minnelide is water-soluble and safe for use in animals.11,13
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We hypothesized that combining sorafenib and triptolide (Minnelide for in vivo experiments) would maximize therapeutic effect and limit toxicity of either drug treatment alone. We showed here that a combination of these drugs at doses that are ineffective when given alone, is superior to either drug treatment in inducing cell death and committing HCC cells to apoptosis in vitro. A distinct advantage of using a combinational therapy is decreasing the dose of sorafenib, where we used 1.25–2.5 mM in our in vitro assays, which corresponds to 1025% of the therapeutic plasma level of an patient with HCC receiving sorafenib at a therapeutic dose of 400 mg twice daily orally.26 At the cellular level, Triptoldie inhibited the prosurvival transcription factor NF-kB in tumor cells. Many investigators suggest that NF-kB activation plays a pivotal role in the development of numerous malignancies by enhancing the local tumoral milieu supply of inflammatory mediators and maintaining chronic inflammatory status.34,35 NF-kB signaling is essential for many cellular functions, however, when constitutively activated in tumor cells, NF-kB signaling enhances the antiapoptotic protein levels as well as proangiogenic pathways.36,37 Also, this change in local tumor environment is believed to transform host innate immune components, such as macrophages, to defend and protect the tumors by evading cellular immune surveillance.34,35 We found that triptolide, but not sorafenib, inhibited basal NF-kB activity, and this effect carried forward into the combination treatment in vitro. Our in vivo data projects an even more compelling story that advocates the use of combinational therapy with sorafenib and Minnelide. We used a low dose of sorafenib (10 mg/kg, which is equivalent to 1.4 mM in vitro concentration26) and show that it induces a modest decrease in tumor volume and proliferation that validates the data published by the developers of sorafenib in 2006.9 More interestingly, we showed that a relatively low dose of Minnelide (0.21 mg/kg) produced the similar decrease in tumor volume and growth. When both drugs were combined, the results were well beyond the additive effect of either drug influence alone, which suggest a synergistic effect. This effect is observed especially in the inhibition of NF-kB binding in tumor xenograft samples. Although neither drug alone decreased p50 transcription factor binding in vivo, the use of combination therapy decreased the binding by 68%. We hypothesize that the use of two drugs that act on different pathways enhances antitumoral because they act in a complementary fashion
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Fig 8. Hypothesis. Sorafenib inhibits the RAF/MEK/ERK signaling pathway, whereas triptolide (Minnelide) inhibits the Akt/mTOR pathway and basal NF-kB activity/activation. Combined together, the two drugs act on different pathways to enhance antitumoral effects.
(Fig 8). Although sorafenib inhibits the RAF/MEK/ ERK signaling pathway, triptolide (Minnelide) inhibited NF-kB activation in a complementary fashion. Triptolide also decreased the mRNA levels of multiple tyrosine kinase receptors in vitro (Supplementary Fig 1; online version only). Most interestingly, we were able to show that combinational therapy is effective in limiting tumor progression over the period of 4-week treatment. It’s difficult to discern why combination treated tumors continued to grow, albeit at a very slow rate, whereas the crossover treated tumors had plateaued during period of treatment (Fig 7). This observation was especially true in a subcutaneous heterotopic tumor model. One possibility is that crossover-treated tumors reached a critical volume at which the treatment-induced decrease in vessel density did not allow further tumor growth, while the combination treated tumors volumes were initially less. This finding could also be a cell-specific phenomenon. To answer this question, we also carried out a similar experiment wherein 36 mice were implanted with PLC/PRF/5-derived tumors and divided into four groups (n = 8); saline, sorafenib (10 mg/kg), Minnelide (0.21 mg/kg), and combination. Combination treatment induced tumor regression to 30% of original tumor size, whereas Minnelide and sorafenib slowed tumor progression to 1.3 ± 0.5- and 3.2 ± 1.1-fold compared with 5.9 ± 3-fold growth in the saline group. When crossed over to combination treatment, saline group tumors regressed to 3 ± 1.4-fold over a 24-day of treatment from 5.9 ± 3 fold; an overall
49% decrease in tumor volumes (Supplementary Fig 2; online version only). Our crossover experiment suggests that the combination of sorafenib and Minnelide not only prevented tumor progression but also caused tumor regression, which is most relevant because it mimics locoregionally metastatic disease. SUPPLEMENTARY DATA Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.surg.2014. 04.055. REFERENCES 1. Forner A, Llovet JM, Bruix J. Hepatocellular carcinoma. Lancet 2012;379:1245-55. 2. Finn RS. Current and future treatment strategies for patients with advanced hepatocellular carcinoma: role of mTOR inhibition. Liver Cancer 2012;1:247-56. 3. El-Serag HB, Davila JA, Petersen NJ, McGlynn KA. The continuing increase in the incidence of hepatocellular carcinoma in the United States: an update. Ann Intern Med 2003;139:817-23. 4. American Cancer Society. Liver Cancer. Chicago, IL: American Cancer Society; 2012. 5. Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, et al. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 2008;359:378-90. 6. Salem R, Hunter RD. Yttrium-90 microspheres for the treatment of hepatocellular carcinoma: a review. Int J Radiat Oncol Biol Phys 2006;66:S83-8. 7. Gedaly R, Angulo P, Hundley J, Daily MF, Chen C, Evers BM. PKI-587 and sorafenib targeting PI3K/AKT/mTOR and Ras/Raf/MAPK pathways synergistically inhibit HCC cell proliferation. J Surg Res 2012;176:542-8. 8. Tanaka S, Arii S. Molecular targeted therapies in hepatocellular carcinoma. Semin Oncol 2012;39:486-92.
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9. Liu L, Cao Y, Chen C, Zhang X, McNabola A, Wilkie D, et al. Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5. Cancer Res 2006;66:11851-8. 10. Shi YH, Ding ZB, Zhou J, Hui B, Shi GM, Ke AW, et al. Targeting autophagy enhances sorafenib lethality for hepatocellular carcinoma via ER stress-related apoptosis. Autophagy 2011;7:1159-72. 11. Phillips PA, Dudeja V, McCarroll JA, Borja-Cacho D, Dawra RK, Grizzle WE, et al. Triptolide induces pancreatic cancer cell death via inhibition of heat shock protein 70. Cancer Res 2007;67:9407-16. 12. Aghdassi A, Phillips P, Dudeja V, Dhaulakhandi D, Sharif R, Dawra R, et al. Heat shock protein 70 increases tumorigenicity and inhibits apoptosis in pancreatic adenocarcinoma. Cancer Res 2007;67:616-25. 13. Chugh R, Sangwan V, Patil SP, Dudeja V, Dawra RK, Banerjee S, et al. A preclinical evaluation of minnelide as a therapeutic agent against pancreatic cancer. Sci Transl Med 2012;4:156ra39. 14. Nagai T, Arao T, Furuta K, Sakai K, Kudo K, Kaneda H, et al. Sorafenib inhibits the hepatocyte growth factor-mediated epithelial mesenchymal transition in hepatocellular carcinoma. Mol Cancer Ther 2011;10:169-77. 15. World Health Organization. Cancer. Available from http:// www.who.int/mediacentre/factsheets/fs297/en/. 16. American Cancer Society. Cancer figures & facts 2013. Chicago: American Cancer Society; 2013. 17. National Cancer Institute. Surveillence, Epidemiology, and End Results Program. Available from: http://seer.cancer. gov/statfacts/html/livibd.html. 18. Wong CM, Ng IO. Molecular pathogenesis of hepatocellular carcinoma. Liver Int 2008;28:160-74. 19. Llovet JM, Bruix J. Molecular targeted therapies in hepatocellular carcinoma. Hepatology 2008;48:1312-27. 20. Huynh H. Molecularly targeted therapy in hepatocellular carcinoma. Biochem Pharmacol 2010;80:550-60. 21. Wilhelm SM, Carter C, Tang L, Wilkie D, McNabola A, Rong H, et al. BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer Res 2004;64:7099-109. 22. Xie B, Wang DH, Spechler SJ. Sorafenib for treatment of hepatocellular carcinoma: a systematic review. Dig Dis Sci 2012;57:1122-9. 23. Chen KF, Tai WT, Liu TH, Huang HP, Lin YC, Shiau CW, et al. Sorafenib overcomes TRAIL resistance of hepatocellular carcinoma cells through the inhibition of STAT3. Clin Cancer Res 2010;16:5189-99. 24. Gedaly R, Angulo P, Hundley J, Daily MF, Chen C, Koch A, et al. PI-103 and sorafenib inhibit hepatocellular carcinoma cell proliferation by blocking Ras/Raf/MAPK and
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
PI3K/AKT/mTOR pathways. Anticancer Res 2010;30: 4951-8. Hikita H, Takehara T, Shimizu S, Kodama T, Shigekawa M, Iwase K, et al. The Bcl-xL inhibitor, ABT-737, efficiently induces apoptosis and suppresses growth of hepatoma cells in combination with sorafenib. Hepatology 2010;52: 1310-21. Hu S, Niu H, Inaba H, Orwick S, Rose C, Panetta JC, et al. Activity of the multikinase inhibitor sorafenib in combination with cytarabine in acute myeloid leukemia. J Natl Cancer Inst 2011;103:893-905. Clawson KA, Borja-Cacho D, Antonoff MB, Saluja AK, Vickers SM. Triptolide and TRAIL combination enhances apoptosis in cholangiocarcinoma. J Surg Res 2010;163: 244-9. Mujumdar N, Mackenzie TN, Dudeja V, Chugh R, Antonoff MB, Borja-Cacho D, et al. Triptolide induces cell death in pancreatic cancer cells by apoptotic and autophagic pathways. Gastroenterology 2010;139:598-608. Dudeja V, Chugh RK, Sangwan V, Skube SJ, Mujumdar NR, Antonoff MB, et al. Prosurvival role of heat shock factor 1 in the pathogenesis of pancreatobiliary tumors. Am J Physiol Gastrointest Liver Physiol 2011; 300:G948-55. Banerjee S, Thayanithy V, Sangwan V, Mackenzie TN, Saluja AK, Subramanian S. Minnelide reduces tumor burden in preclinical models of osteosarcoma. Cancer Lett 2013;335: 412-20. Rousalova I, Banerjee S, Sangwan V, Evenson K, McCauley JA, Kratzke R, et al. Minnelide: a novel therapeutic that promotes apoptosis in non-small cell lung carcinoma in vivo. PLoS ONE 2013;8:e77411. Antonoff MB, Chugh R, Borja-Cacho D, Dudeja V, Clawson KA, Skube SJ, et al. Triptolide therapy for neuroblastoma decreases cell viability in vitro and inhibits tumor growth in vivo. Surgery 2009;146:282-90. Krosch TC, Sangwan V, Banerjee S, Mujumdar N, Dudeja V, Saluja AK, et al. Triptolide-mediated cell death in neuroblastoma occurs by both apoptosis and autophagy pathways and results in inhibition of nuclear factor-kappa B activity. Am J Surg 2013;205:387-96. Beyaert R, Beaugerie L, Van Assche G, Brochez L, Renauld JC, Viguier M, et al. Cancer risk in immune-mediated inflammatory diseases (IMID). Mol Cancer 2013;12:98. Ben-Neriah Y, Karin M. Inflammation meets cancer, with NF-kappaB as the matchmaker. Nat Immunol 2011;12: 715-23. Chaturvedi MM, Sung B, Yadav VR, Kannappan R, Aggarwal BB. NF-kappaB addiction and its role in cancer: ‘one size does not fit all’. Oncogene 2011;30:1615-30. Hoesel B, Schmid JA. The complexity of NF-kappaB signaling in inflammation and cancer. Mol Cancer 2013; 12:86.