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There are limited chemotherapy options for hepatocellular carcinoma (HCC). The heparin-degrading endosulfatase SULF1 functions as a liver tumor suppressor. We investigated the effects of the histone deacetylase inhibitor apicidin in combination with doxorubicin in SULF1-expressing HCC cells in vitro and in SULF1-expressing xenografts in nude mice.
We evaluated the effects of apicidin alone or combined with doxorubicin on apoptosis, caspase activity, and phosphorylation of Erk and Akt in SULF1-transfected Huh7 and Hep3B cells in vitro and in vivo.
Apicidin induced HCC cell apoptosis and caspase activation in a dose- and time-dependent manner. Apicidin-induced caspase activation was significantly inhibited by the caspase inhibitor Z-Vad-fmk. Apicidin also decreased phosphorylation of both Erk and Akt. Expression of constitutively-active Mek1 and Akt significantly decreased apicidin-induced apoptosis. The combination of doxorubicin with apicidin significantly increased the anti-tumor effect in the SULF1-expressing Huh7 and Hep3B cells as compared to either apicidin or doxorubicin alone, both in vitro and in vivo.
The combination of a histone deacetylase inhibitor with doxorubicin may be a novel and promising therapeutic modality for HCCs, particularly for SULF1-expressing HCCs.
Hepatocellular carcinoma (HCC) is the third most frequent cause of cancer death worldwide (1). Survival of patients diagnosed with HCC is poor and only 10-20% of HCCs are detected at a sufficiently early stage for radical, potentially curative therapy to be feasible (2). Local therapies for tumor control do not result in durable responses, and there are only limited options for chemotherapy for HCC(3). Therefore, new chemotherapeutic agents or methods to improve the efficacy of existing agents are needed for effective treatment of the majority of HCCs. The recent demonstration of efficacy of sorafenib in advanced HCC provides a rationale for targeted therapies for HCC (4, 5).
The cellular gene expression program is regulated by the level of acetylated histones associated with nuclear chromatin. Pharmacological manipulation of chromatin remodeling by histone deacetylase (HDAC) inhibitors has been proposed as a strategy for treatment of cancer. HDAC inhibitors suppress the activities of multiple HDACs, leading to an increase in histone acetylation. Histone acetylation enhances the expression of genes that elicit cellular growth arrest, differentiation and apoptosis (6). Apicidin is a novel HDAC inhibitor derived from a fungal metabolite (7-9). Its anti-tumor action in HCC remains to be elucidated. Sulfatase 1 (SULF1) is a recently characterized heparin-degrading endosulfatase which is downregulated in 9 out of 11 human HCC cell lines and in approximately 30% of primary HCC’s (10). We have shown that SULF1 desulfates cell surface proteoglycans and prevents the formation of the signaling complex between heparin-binding growth factor ligands, heparan sulfate proteoglycans, and receptor tyrosine kinases at the cell surface. Expression of SULF1 therefore inhibits receptor tyrosine kinase signaling and abrogates cellular growth and survival signaling, thus sensitizing cells to chemotherapy-induced apoptosis (10-13). We have found that SULF1 increases histone H4 acetylation in HCC cell lines and potentiates the therapeutic effect of the HDAC inhibitors apicidin and scriptaid in these cells (7). SULF1 also functions as a tumor suppressor in cancers of the breast (14-16), pancreas (13, 17), ovary (11, 18, 19), head and neck (12), and in multiple myeloma (20).
Doxorubicin (DOX) has been widely used for the treatment of HCC (21-23). The anti-tumor effects of DOX are mainly through DNA-damage leading to apoptosis of tumor cells. However, DOX also increases phosphorylation of MAP kinase, which may decrease its anti-tumor efficacy (24). Therefore, it is not surprising that reports of its efficacy are not consistent, with response rates varying up to 20%. Interestingly, early phase studies of the combination of doxorubicin with the multi-kinase inhibitor, sorafenib, suggest that the combination may be more efficacious than either agent alone. Combining DOX with a targeted therapeutic approach may therefore provide additional treatment options (23, 25).
In the present study, we investigated the anti-tumor efficacy of apicidin in HCC in vitro and in vivo, and evaluated the efficacy of the combination of apicidin with DOX in HCC cells with or without expression of SULF1. We addressed the following questions: 1) Is apicidin-induced apoptosis caspase dependent? 2) Does apicidin decrease phosphorylation of Erk and Akt, which are among the most important pathways in development of HCCs? 3) Does apicidin induce apoptosis and inhibit tumor growth of HCC xenografts? 4) Does the combination of apicidin with DOX increase caspase activation and anti-tumor effects in SULF1-expressing HCC cells in vitro? and 5) Does the combination of apicidin with DOX show increased anti-tumor effects in SULF1-expressing HCC xenografts in vivo?
Apicidin was purchased from Calbiochem (Cambridge, MA); SB, TSA, 4, 6-diamidino-2-phenylindole dihydrochloride (DAPI) was obtained from Sigma (St Louis, MO); DOXIL (doxorubicin HCL liposome) was purchased from Ortho Biotech, Raritan, NJ; polyclonal antibodies against human acetylated histone H3, acetylated histone H4, phospho-ERK44/42, total ERK44/42, phospho-AKT ser 473 and total AKT were purchased from Cell Signaling (Beverly, MA); monoclonal human anti-procaspase 9 was purchased from Pharmingen (Boston, MA); anti-actin antibody and horseradish peroxidase-conjugated mouse IgG and rabbit IgG were from Santa Cruz Biotechnology (Santa Cruz, CA); ECL reagents were purchased from Pierce (Rockford, IL). Apo-One Homogeneous Caspase 3/7 Assay kits were purchased from Promega (Madison, WI).
The three HCC cell lines, Hep3B, SNU449 and SNU182 were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured as recommended by ATCC. Huh7 was from Dr. Gregory Gores’ lab. Cells were maintained in a 37 °C, 5% CO2 incubator, and in logarithmic growth.
Fluorescence microscopy: Apoptosis of Huh7, Hep3B, SNU449 or SNU182 in vitro was measured by identification of apoptotic nuclear changes (chromatin condensation and nuclear fragmentation) using fluorescence microscopy after staining with the DNA binding dye, DAPI (7). Three hundred cells were randomly counted, and each treatment was repeated at least three times, in two wells each time.
The identification of apoptotic cells by morphological criteria using light-microscopic examination of haematoxylin–eosin (H&E)-stained sections is generally considered to be the reference standard in paraffin embedded tissues (26-30). Morphological characteristics including the presence of apoptotic bodies, nuclear condensation and cytoplasmic shrinkage were assessed in H&E-stained tissue sections of the xenografts. In all cases, at least 500 tumor cells in 6 randomly-selected high power fields/slide were counted and morphologically apoptotic cells were expressed as a percentage of the total number of cells counted (apoptotic index) (29, 31, 32).
To determine whether the combination of apicidin with DOX enhances activation of caspases −3 and −7, the activity of caspases-3 and -7 in 2μg of protein from whole cell lysates of HCC cells was measured (7).
Twenty micrograms of total cell protein were separated by electrophoresis (7, 10). Blots were probed with rabbit polyclonal anti-acetylated histone H3 and H4, mouse monoclonal anti-procaspase 9, rabbit polyclonal anti-phospho-Erk and anti-total Erk, and rabbit polyclonal anti-phospho-Akt ser473 and anti-total Akt. The level of Actin was also measured to control for equal loading.
Plasmids encoding enhanced GFP under the transcriptional control of the human cytomegalovirus immediate-early promoter (pEGFP) and hemagglutinin-tagged activated Mek1 and Akt were obtained from Upstate Biotechnology. The entire Mek1 cDNA was subcloned into plasmid vector pEGFP. Log-phase Huh7 and Hep3B cells were transfected using a mixture of Mek1-expressing plasmid DNA or pEGFP-C2 vector DNA and lipofectamine Plus reagent (Invitrogen). Similarly, constitutive Akt and related control vector were transfected into Huh7 cells.
SULF1 expressing and empty vector plasmids were stably transfected into the SULF1 negative HCC cell lines Huh7 and Hep3B as described previously(7, 10). The anti-tumor efficacy of the combination of apicidin with DOX was evaluated using the MTT assay(7, 10).
Huh7 and Hep3B cells with or without SULF1 expression (1×106) were suspended in PBS and inoculated subcutaneously into the right flanks of 4- to 6-week-old male BALB/c nu/nu nude mice (NCI/NIH). Tumor sizes were measured with calipers and when the tumor size reached 400 to 800 mm3, the mice were randomly grouped (4 mice in each group) and treated by intraperitoneal injection of apicidin at a dose of 2.5 mg/kg body weight or with control 1% DMSO. The treatment was performed daily for one week, every other day for the second week and then every three days for the subsequent four weeks. Doxil (DOX) at a dose of 3mg/kg body weight was intravenously injected via tail vein every 6 days for 4 times. Tumors were measured every three days with calipers and tumor volumes (TVs) calculated using the formula: TV=LS2/2 (where L is the longest diameter and S is the shortest diameter).
The experiment with 4 mice in each group was repeated. A piece of tissue from each xenograft was fixed in formalin and paraffin embedded. Paraffin sections were then stained with hematoxylin and eosin. Histologic examination was carried out by light microscopy using a Leica DM LB microscope (Leica Microsystems, Inc., Bannockburn, IL). For quantitation of apoptosis in the H&E sections (C and D), the percent apoptosis in 500 nuclei from 6 randomly selected areas of each slide was counted(29, 31, 32). All experimental protocols were approved by the Institutional Animal Care and Use Committee (IACUC), Mayo Clinic and Foundation, Rochester, Minnesota.
All data represent at least 3 independent experiments, each performed in duplicate or triplicate using cells from separate cultures and are expressed as the mean ± SEM. Differences between groups were compared using Student’s test.
We evaluated apoptosis induced by apicidin in four HCC cell lines: Huh7, SNU449, SNU182 and Hep 3B. Cells were treated with 0 to 5 μM of apicidin for 24 to 72 hours. Apicidin induced apoptosis in all four cell lines. Representative figures showing condensed and fragmented apoptotic nuclei in Huh7 cells after apicidin treatment are shown in Figure 1A. Similar results were obtained for SNU449, SNU182 and Hep 3B cells (data not shown). Apicidin induced apoptosis in the Huh7 and Hep3B cells in a dose- and time-dependent fashion (Figure 1B and 1C).
To determine whether apicidin-induced apoptosis requires caspase activation, cells were pretreated with the caspase inhibitor Z-VAD-fmk 1 hour prior to apicidin treatment. Apicidin induced apoptosis was significantly inhibited by 10 μM of Z-VAD-fmk in both Huh7 and Hep3B cells (P<0.01, Figure 1D). Apicidin also increased caspase-3/7 activity in a dose dependent manner (Figure 1E) and these effects were significantly inhibited by Z-VAD-fmk (P<0.05, Figure 1F).
Previously we have shown that the level of acetylated histone H4 was detectable but low in all six HCC cell lines tested, including Huh7 and Hep3B cells(7). Caspase 9 activation is an early marker of caspase-mediated apoptosis. It has been reported that HDAC inhibitors induce cancer cell apoptosis via mitochondrial dependent activation of the caspase cascades. To compare the time course of activation of caspase 9 with that of histone acetylation, Huh7 cells in regular media 10% FBS were treated with apicidin for 24 hours, and then harvested. Western blotting was performed using antibodies against procaspase 9 and acetylated histone H4. At 24 hours after treatment with apicidin, acetylated histone H4 increased significantly in Huh7 cells, and cleavage of a small amount of pro-caspase 9 was seen at higher doses (Figure 2A). Therefore HDAC inhibitor induced histone acetylation also appears to precede caspase 9 activation.
The MAPK and PI3 kinase/Akt kinase pathways are important contributors to growth and survival of HCC cells. Since growth of HCCs is determined by the balance between the effects of pro-survival and pro-apoptotic pathways, we investigated the changes in MAPK and Akt kinase activation during the process of HDAC inhibitor-induced apoptosis. For these experiments, the blots shown in Figure 2A were stripped and re-probed with anti-phospho-Erk44/42 and anti-phospho-Akt ser473 antibodies. The Western blotting results showed that phosphorylation of both Erk44/42 and Akt was down regulated by low concentrations of the HDAC inhibitor apicidin in Huh7 (Figure 2A), Hep3B and SNU449 cells (data not shown).
We have found that both Erk44/42 and PI3 kinase inhibitors decrease the viability of HCC cells (Lai, et al, unpublished data). To determine whether the Erk44/42 or Akt pathways are involved in apoptosis induced by HDAC inhibitors, we examined the effects of Erk44/42 or PI3 kinase inhibitors on HCC cell apoptosis. Huh7 cells were treated with 25 μM of either the Erk44/42 inhibitor U0126 or the PI3 kinase inhibitor LY294002 in the presence or absence of apicidin. We found that LY294002 significantly induced apoptosis of Huh7 cells (mean percent apoptosis 1.8% vs. 38.8%, P<0.01) and also enhanced apicidin-induced apoptosis (27.2% vs. 65.7%, P<0.01). By contrast, the Erk inhibitor U0126 showed only a relatively small induction in apoptosis (1.8% vs. 4.9%) and therefore was not as potent in enhancing apicidin-induced apoptosis (data not shown). To determine whether activated Erk or Akt block apicidin-induced apoptosis, we transiently transfected constructs expressing constitutively active GFP-Mek1 or Akt into Huh7 and Hep3B cells; and the related empty vector GFP construct was used as the control. Apoptosis was evaluated at 24 and 48 hours after treatment with 2.5 μM of apicidin. We found that both constitutively active Mek1 and Akt significantly attenuated apicidin-induced apoptosis (P<0.05) (Figure 2B and 2C). The results showing that 25 μM U0126 minimally induces apoptosis but constitutive activation of Mek1 significantly decreases apicidin-induced apoptosis in HCC cells are consistent with the reported effects of U0126 and Mek1 activation in sodium butyrate (SB) and SAHA-induced apoptosis in human leukemia K562 cells(33).
Previously we have already shown that apicidin inhibits tumor growth in part by inhibition of tumor angiogenesis(7). To investigate the mechanism of inhibition of apicidin in HCC tumor growth in vivo, we repeated the treatment of mice bearing Huh7 xenografts with apicidin and sacrificed both control and apicidin treated mice on the 15th day after initiation of apicidin or DMSO treatment. Tissue from the xenografts was fixed in formalin and paraffin embedded. After H&E staining, all sections were evaluated for cancer cell apoptosis by light microscopy. Apoptotic nuclei were counted in 6 different regions in both control and apicidin-treated mice. Treatment with apicidin significantly increased tumor cell apoptosis in Huh7 xenografts in vivo as compared to the DMSO control (Figure 3A, B and C).
To determine the efficacy of the combination of apicidin with DOX in HCC cells with or without expression of SULF1, we treated the Huh7 and Hep3B cells stably expressing SULF1 with apicidin, DOX, or the combination and evaluated cell viability by the MTT assay and apoptosis by DAPI staining and fluorescence microscopy. We found that either apicidin or DOX alone significantly decreased viability of SULF1 expressing Huh7 cells (P<0.05), but not of the vector-transfected Huh7 cells (P>0.05). Cell viability was further significantly decreased by the combination of the two agents (P<0.001) (Figure 4A). Similar results were also found for SULF1 expressing Hep3B cells (data not shown). Either apicidin or DOX induced apoptosis in vector-transfected Huh7 cells, and these effects were significantly increased by SULF1 expression in two SULF1-expressing Huh7 clones (P<0.01) and enhanced by the combination of both agents (P<0.001) (Figure 4B). To confirm these effects in Hep3B cells, we repeated the experiments in a vector transfected Hep3B clone and two SULF1-expressing Hep3B clones. Enhanced efficacy was also found in SULF1-expressing Hep3B clones (P<0.001), but not in the vector transfected Hep3B cells (Figure 4C and 4D).
Since DOX has been shown to activate Erk phosphorylation, we determined the levels of p-Erk and total Erk after treatment with either apicidin or DOX alone or apicidin combined with DOX. Compared to the baseline p-Erk in cells treated with control DMSO, apicidin decreased p-Erk as reported, but DOX increased Erk. The combination of apicidin and DOX abolished the DOX-induced increase in p-Erk in Hep3B cells, particularly in SULF1 expressing Hep3B cells (Figure 4E and 4F). We also repeated these experiments in Huh7 cells and similar results were obtained (data not shown). To evaluate the effects of apicidin and DOX on caspase activation, we collected cell pellets 48 hours after treatment and measured caspase 3 and 7 activity in the whole cell lysates from Hep3B Vector and SULF1-expressing clones. The combination of apicidin with DOX significantly increased activation of caspases 3 and 7 (P<0.0001) (Figure 4G).
Previously we have shown that apicidin inhibited tumor growth of both Huh7 and Hep3B xenografts(7). Here, we compared the anti-tumor effects of DOX alone and in combination with apicidin on Huh7 SULF1-expressing xenografts and Huh7 vector xenografts. SULF1 expression in the xenografts (Figure 5A and B) was also confirmed by immunohistochemistry after the mice were sacrificed. We found DOX inhibited growth of Huh7 vector xenografts as compared to the DMSO control, but these effects were not statistically significantly enhanced by the combination of DOX with apicidin (Figure 5C and 5E). Compared to the Huh7 vector cell xenografts, Huh7 SULF1 expressing xenografts showed slower tumor growth(7), and also demonstrated significantly enhanced responses to apicidin or DOX (P<0.05). Further, in SULF1-expressing Huh7 xenografts, the combination of apicidin with DOX significantly enhanced anti-tumor effects as compared with either apicidin and DOX alone (P<0.05) (Figure 5D and 5F). Similar results were also found in SULF1-expressing Hep3B xenografts (data not shown).
Chemotherapy options for advanced hepatocellular carcinoma remain limited, with sorafenib being the only approved agent. We are currently investigating the modulation of new pathways for the treatment of hepatocellular carcinoma. In the present study we found that the newly identified HDAC inhibitor, apicidin, induces apoptosis of four different HCC cell lines examined. The effect of apicidin was similar to that of members of two other chemical classes of HDAC inhibitors, SB and TSA (Lai, et al, unpublished data). As expected, the effect of the HDAC inhibitors was associated with increases in acetylation of nucleosomal histones H3 and H4 (Lai, et al, unpublished data). Apicidin-induced apoptosis was associated with downregulation of MAP kinase and Akt kinase activity. Further, we found that apicidin not only induces apoptosis in HCC cells in vitro, but also induces HCC cell apoptosis in vivo and inhibits angiogenesis in nude mouse xenografts. In addition, combination of apicidin with doxorubicin resulted in additive increases in both caspase activation and anti-tumor activity in SULF1-expressing HCC cells both in vitro and in vivo.
Apicidin is a novel HDAC inhibitor with a potent broad spectrum of antiproliferative activity against multiple cancer cell lines(34-36). Apicidin has been shown to induce HL-60 leukemia cell apoptosis through the selective induction of the Fas/Fas ligand pathway, resulting in the subsequent activation of caspase 9 and caspase 3 (37). In the present study, we have shown that apicidin induces apoptosis in all four HCC cell lines examined, Huh7, Hep3B, SNU182 and SNU449, in both a time- and dose-dependent manner. We show that the induction of apoptosis of HCC cell lines by apicidin occurs through a caspase-dependent mechanism. Somewhat surprisingly, the caspase inhibitor Z-VAD-fmk not only blocks apicidin-induced apoptosis, but also inhibits histone H4 acetylation in both Huh7 and SNU182 cells (data not shown). This suggests a potential effect of caspase activation on histone acetylation and needs to be explored further. Although it is presumed that the actions of HDAC inhibitors result from histone acetylation and transcriptional activation of genes involved in these processes, the specific downstream events responsible for induction of cell death remain to be fully elucidated.
We have previously shown that SULF1 inhibits HCC cell proliferation in vitro and in vivo through down-regulation of heparin-binding growth factor signaling and also partly through acetylation of histone H4. Consequently, SULF1 potentiates the therapeutic efficacy of the histone deacetylase inhibitors apicidin and scriptaid(7). The growth of HCCs depends on the balance between cell proliferation/survival pathways and cell death-promoting pathways. MAPK activation leads to phosphorylation of Erk44/42 (Erk1/2), and sustained dual phosphorylation of Erk44/42 is required for HCC cell proliferation (10, 11). The Ras/Raf/mitogen-activated protein/extracellular signal-regulated kinase pathway is one of the most critical signaling cascades for liver tumorigenesis(24, 38). Ligand independent constitutive activation of the Ras/Raf/Mek/Erk pathway can play an important role in tumor development and is considered another mechanism of resistance to receptor-targeted therapy (39). Activation of the PI3 kinase/Akt kinase pathway also inhibits cell death and promotes cell survival (40), and recent studies suggest that inactivation of this cascade sensitizes neoplastic cells to drug-induced apoptosis(10, 41). In the present study we found that constitutive activation of both Mek and Akt diminished apicidin-induced apoptosis of tumor cells. This may explain why we did not observe a synergistic effect of the combination of apicidin with DOX in SULF1-negative HCC xenografts. Although, DOX has been one of the most widely used chemotherapeutic agents in HCC, Huyhn et al reported that a low dose of DOX (0.17 μM in vitro, 1 mg/kg in vivo) increased Erk phosphorylation(24). In HCC, multiple molecular alterations ensure the progressive growth of tumor cells. Rapid tumor growth is closely linked to chemotherapy resistance(42). In the present study, we confirmed that DOX alone activated Erk in Hep3B and Huh7 cells, however, either expression of SULF1 or treatment with apicidin inactivated Erk in the cells. Consequently, the combination of apicidin with DOX abolished the DOX-induced activation of Erk and enhanced caspase activation and anti-tumor activity in SULF1-expressing HCC xenografts. These findings may lead to new strategies for treatment of HCCs.
In summary, we have made the novel observations that the HDAC inhibitor apicidin induces caspase activation and Erk and Akt inactivation, thus promoting apoptosis and inhibiting tumor growth of HCCs. The combination of apicidin with doxorubicin enhances the antitumor effects of doxorubicin on caspase activation and tumor growth in SULF1-expressing HCCs. Future translation of these effects may lead to development of novel therapeutic modalities for human HCCs.
This work was supported by Mayo Clinic and Mayo Cancer Center and by NIH Grants CA82862 and CA100882, an Industry Research Scholar Award from the Foundation for Digestive Health and Nutrition, a Harold Amos Medical Faculty Development Award from The Robert Wood Johnson Foundation and by the Miles and Shirley Fiterman Center for Digestive Diseases at the Mayo Clinic, Rochester, MN (to L.R.R.).
The authors thank Vicki Campion and Erin Nystuen-Bungum for secretarial assistance and Dr. Gregory Gores for his critical review of the manuscript.
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