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The anti-tumor activity of histone deacetylase inhibitors (HDACI) on multi-drug resistant sarcoma cell lines has never been previously described. Four multidrug resistant sarcoma cell lines treated with HDACI PCI-24781 resulted in dose-dependent accumulation of acetylated histones, p21 and PARP cleavage products. Growth of these cell lines was inhibited by PCI-24781 at IC50 of 0.43 to 2.7. When we looked for synergy of PCI-24781 with chemotherapeutic agents, we found that PCI-24781 reverses drug resistance in all four multidrug resistant sarcoma cell lines and synergizes with chemotherapeutic agents to enhance caspase-3/7 activity. Expression of RAD51 (a marker for DNA double-strand break repair) was inhibited and the expression of GADD45α (a marker for growth arrest and DNA-damage) was induced by PCI-24781 in multidrug resistant sarcoma cell lines. In conclusion, HDACI PCI-24781 synergizes with chemotherapeutic drugs to induce apoptosis and reverses drug resistance in multidrug resistant sarcoma cell lines.
Sarcomas represent a heterogeneous group of connective tissue-derived tumors comprising more then 50 histologic subtypes1. There will be approximately 13,000 new cases of sarcomas diagnosed this year in the United States, and 5,000 deaths2. Available therapies for advanced sarcomas include chemotherapy, surgery, and radiotherapy. However, development of drug resistance is a major barrier to successful treatment as most patients either do not respond to chemotherapy or eventually develop resistance3, 4. The overall 5-year survival rate in patients with soft tissue sarcomas of all stages remains poor, at only 50–60%5, 6. Use of multimodality treatment approaches is crucial, but current treatment approaches are unable to significantly prolong survival. Several strategies have been attempted to reverse drug resistance in other types of human cancer, including small molecular compounds, siRNA, and nanotechnology7-9. Various chemical agents can restore drug sensitivity in tumor cells, and these possibilities are currently being explored in attempts to develop anticancer therapies. Among such promising agents are histone deacetylase (HDAC) inhibitors.
Histone deacetylases (HDACs) play an important role in the epigenetic regulation of gene expression by catalyzing the removal of acetyl groups, stimulating chromatin condensation, and promoting transcriptional repression10, 11. Since aberrant epigenetic changes are common and significant mechanisms in cancer development and progression, HDACs are promising targets for pharmacological inhibition. HDAC inhibitors (HDACI) can mediate mis-regulation of a number of genes within cancer cells, these include cell cycle regulators mediating G1 arrest, inhibitors of DNA synthesis, apoptosis regulators, and gene expression modulators12-15. These properties have prompted numerous preclinical and clinical investigations evaluating the potential efficacy of HDACI for multiple types of cancers, essentially showing promise as anticancer agents. Although HDACI do show promise as single agents, another potential for HDACIs may lie in their ability to modulate the activity of other therapeutic agents. In cancers that respond poorly to chemotherapy, treatment with HDACI can increase the sensitivity of the cancer cells to other drugs and treatments such as radiotherapy. HDACIs including Vorinostat, depsipeptide, MS-275, and TSA, have been shown to additively or synergistically enhance the anticancer activity of a large number of conventional chemotherapeutic drugs 16-18. These drugs include gemcitabine, paclitaxel, cisplatin, etoposide, VP-16, and doxorubicin, all of which eliminate cancer cells through different mechanisms 16, 19-22. Their broad capacity for synergy indicates that HDACI likely lower the threshold for tumor cells to undergo apoptotic cell death triggered by other agents.
PCI-24781 is a hydroxamic acid–based HDAC inhibitor that was developed on in vivo efficacy and therapeutic index 23. It is currently undergoing testing for safety, tolerability, and pharmacokinetics in several phase I trials. Preclinical results, using various treatment schedules, have established growth inhibitory concentrations for several tumor cell lines, as well as tumor growth inhibition in three xenograft models.23. Tumor cells are thought to be more sensitive than normal cells to both the growth inhibiting and apoptosis promoting effects of most HDACIs. Microarray analysis with PCI-24781-treated cells has confirmed up-regulation of p21 and caspases and down-regulation of cyclins23. While increases in DNA accessibility caused by changes in acetylation may also enhance DNA damage and repair the damages more directly 24, 25, the efficacy of PCI-24781 on mutidrug resistant sarcoma cells has not been reported before.
In the present study, we investigated the anti-tumor activity of PCI-2478 on multi-drug resistant sarcoma cell lines. We observed that PCI-24781 induces apoptosis and inhibits growth of multi-drug resistant sarcoma cells. Furthermore, PCI-24781 significantly enhanced the apoptotic cell killing effect of chemotherapeutic drugs and reverses drug resistance in multi-drug resistant sarcoma cells.
Human osteosarcoma cell line U-2 OS was obtained from the American Type Tissue Collection (Rockville, MD). The Multidrug resistant cell line U-2 OS MR was established by the continuous culture of U-2 OS in medium containing step-wise increases in paclitaxel concentration over a period of 8 months26. Dr. Efstathios S. Gonos (National Hellenic Research Foundation, Athens, Greece)27 kindly provided the human multidrug resistant cell line KH OS R2, which were derived from the KH OS parent cell line by continuous exposure to doxorubicin. Dr. Katia Scotlandi (Institute Orthopedics Rizzoli, Italy) kindly provided the multidrug resistant Ewing’s sarcoma cell line TC-ET, which was derived from the TC-71 parent cell line by continuous exposure to ET-74328. The multidrug resistant chordrosarcoma cell line CS-ZR established in our laboratory was derived from the CS-1 parent cell line by continuous exposure to Zalypsis (PM00104). All cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin (all obtained from Invitrogen, Carlsbad, CA.). Resistant cell lines were continuously cultured in chemotherapeutic drugs. HDAC inhibitor PCI-24781 was synthesized and provided by Pharmacyclics, Inc. Doxorubicin was obtained as unused clinical material at the Massachusetts General Hospital. ET-743 and Zalypsis were supplied by PharmaMar (Spain).
Protein lysates from cells were generated through lysis with 1×RIPA Lysis Buffer (Upstate Biotechnology, Charlottesville, VA). The concentration of the protein was determined by Protein Assay Reagents (Bio-Rad, Hercules, CA) and spectrophotometer (Beckman DU-640, Beckman Instruments, Inc., Columbia, MD). Forty micrograms of total protein was processed on Nu-Page 4-12% Bis-Tris Gel (Invitrogen) and transferred to a pure nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). Antibodies directed against acetylated histones were obtained from Millipore Corporate (Billerica, MA). Antibodies directed against PARP were obtained from Cell Signaling Technologies (Cambridge, MA). Antibodies directed against p21 were obtained from BD Biosciences (San Jose, CA). Antibodies directed against RAD51, GADD45α and actin were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). Primary antibodies were incubated at 1:1000 dilution in Tris-buffered saline, pH 7.4, with 0.1% Tween 20 and overnight at 4°C. Signal was generated through incubation with horseradish peroxidase-conjugated secondary antibodies (Bio-Rad, Hercules, CA) incubated in Tris-buffered saline, pH 7.4, with 5% nonfat milk and 0.1% Tween 20 at 1:2000 dilution for 1 h at room temperature. Positive immunoreactions were detected by using SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL).
The in vitro cytotoxicity assays were performed by MTT assay as previously described26. MTT was obtained from Sigma (St. Louis, MO). 1.5×103 cells per well were plated in 96–well plates of DMEM medium containing PCI-24781 and/or chemotherapeutic drugs. For the cells treated with the combination of PCI-24781 and chemotherapeutic drugs, the cells were treated with PCI-24781 for 4 h at first, then the chemotherapeutic drugs were added. After culture in PCI-24781 and/or chemotherapeutic drugs for 7 days, 10μL of MTT (5mg/ml in PBS) was added to each well and the plates were incubated for four hours. The resulting formazan product was dissolved with acid-isopropanol and the absorbance at a wavelength of 490nm (A490) was read on a BT 2000 Microkinetics Reader. Experiments were performed in duplicate.
Caspase activity was measured with the Apo-One Homogeneous Caspase 3/7 assay kit (Promega Corporation), according to the manufacturer’s instructions. The induction of apoptosis and associated activation of caspases 3 and 7 are measured by enzymatic cleavage of the profluorescent substrate rhodamine 110, bis-N-CBZ-L-aspartyl-L-glutaml-L-valyl-L-aspartic acid amide (Z-DEVD-R110), with release of the intensely fluorescent rhodamine 110-cleaving group. Cells were seeded at a density of 1×105/ml and incubated in a 96-well plate in the presence or absence of drug for 48 h. For cells treated with a combination of PCI-24781 and chemotherapeutic drugs, they were initially treated with PCI-24781 for 4 h prior to the addition of chemotherapeutic drugs. 100 μl of the homogeneous caspase-3/7 reagent was added to each well and reaction mixture was incubated for 2 h at room temperature, and then fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm. Results are expressed as relative fluorescence units (RFU).
Values shown are representative of triplicate determinations in two or more experiments. Treatment effects were evaluated using a two-sided Student’s t test (GraphPad PRISM® 4 software, GraphPad Software, San Diego, CA). Errors are SD of averaged results and P< 0.05 values were accepted as a significant difference between means.
In order to determine if HDAC inhibition by PCI-24781 affects the proliferation of sarcoma cells, a panel of drug resistant sarcoma cell lines and their parental cell lines, including multidrug resistant osteosarcoma cell line U-2 OS MR and the parental cell line U-2 OS, multidrug resistant osteosarcoma cell line KH OS R2 and the parental cell line KH OS, multidrug resistant chorndrosarcoma cell line CS-ZR and the parental cell line CS-1, and multidrug resistant Ewing’s sarcoma cell line TC-ET and the parental cell line TC-71, were treated in vitro with various concentrations of PCI-24781. The accumulation of several mechanistic biomarkers proposed to be involved in the antitumor activity of HDAC inhibitors was analyzed by Western blot after treatment with PCI-24781. The growth of sarcoma cells was evaluated by MTT after treatment with PCI-24781.
Western blot analysis demonstrated that treating drug resistant sarcoma cell lines with PCI-24781 resulted in the dose-dependent accumulation of acetylated histones (Fig. 1), indicating that HDAC enzymes are inhibited in these cells. In addition, PCI-24781 induced expression of the cyclin-dependent kinase inhibitor, p21, a protein postulated to play a role in the antitumor effect of HDAC inhibition.
MTT assay demonstrated that the growth of all four multidrug resistant sarcoma cell lines was inhibited after treatment with PCI-24781 at IC50 from 0.43 to 2.7. The parental osteosarcoma cell line KH OS, chorndrosarcoma cell line CS-1 and Ewing’s sarcoma cell line TC-71 are significantly sensitive to PCI-24781 compared to their multidrug resistant cell lines(Fig. 2). The effect of PCI-24781 on induction of apoptosis was determined by Western blot for PARP cleavage, an apoptotic marker associated with apoptosis biochemical event. Accumulation of PARP cleavage was detected in all four multidrug resistant sarcoma cell lines after treatment with PCI-24781(Fig. 1).
To evaluate if PCI-24781 enhances the anticancer activity of conventional chemotherapeutic drugs in multidrug resistant sarcoma cells, multidrug resistant sarcoma cell lines were coincubated with PCI-24781 and a sub-lethal concentrations of one of several chemotherapeutic drugs. The drugs examined included DNA-damaging agents such as doxorubicin, ET-743, and Zalypsis. Cytotoxicity assay demonstrated that the multidrug resistant cells, U-2 OS MR, KH OS R2, CS-ZR and TC-ET, underwent greater growth inhibition to doxorubicin, Zalypsis, and ET 743 when coincubated with PCI-24781 (Fig. 3). The IC50 values for doxorubicin in parental U-2 OS MR cells (IC50=0.172) were 6 fold more than PCI-24871 treated U-2 OS MR cells (IC50=0.027). The IC50 values for doxorubicin in parental KH OS R2 cells (IC50=4.03) were 5 fold more than PCI-24871 treated KH OS R2 cells (IC50=0.8). The IC50 values for zalypsis in parental CS-ZR cells (IC50=0.0056) were 2.4 fold more than PCI-24871 treated CS-ZR cells (IC50=0.0023). The IC50 values for ET 743 in parental TC-ET cells (IC50=2.58) were 1.8 fold more than PCI-24871 treated TC-ET cells (IC50=1.43). The caspase-3/7 activity assay demonstrated an increase in caspase-3/7 activity in the multidrug resistant sarcoma cell lines after co-treatment with both PCI-24781 and chemotherapeutic drugs when compared with the multidrug resistant sarcoma cells treated with PCI-24781 or chemotherapeutic drugs alone (Fig. 4). The same concentration of drugs induced only a small increase in caspase-3/7 activity in multidrug resistant cell lines, however, these effects were significantly enhanced by PCI-24781 (Fig.4)
RAD51 is a homologous recombination (HR) and DNA repair protein. Increased expression RAD51 has been reported in immortalized cell lines and in multiple primary tumor cell types which plays a role in either the initiation or in the progression of tumorigenesis29-33. It has been reported that upregulated RAD51 may lead to resistance of irradiation or cytotoxic agents, impairing protein interactions, altering the fidelity of HR-mediated chromosomal double-strand breaks(DSBs) repair, and gross chromosomal aberrations33.
To investigate the mechanism underlying the reversion of multidrug resistance of multidrug resistant sarcoma cells by PCI-24781, multidrug resistant sarcoma cell lines were treated with various concentrations of PCI-24781. The expression of RAD51 and GADD45α was evaluated by Western blot after treatment with PCI-24781. Western blot demonstrated that the expression of RAD51 was inhibited by PCI-24781 in all four multidrug resistant sarcoma cell lines (Fig. 5). The expression of GADD45α was induced by PCI-24781 in multidrug resistant osteosarcoma cells U-2 OS MR and KH OS R2 (Fig. 5). However, the expression of GADD45α was not increased after treatment with PCI-24781 in CS-ZR cells, while no expression of GADD45α was found in multidrug resistant Ewing’s sarcoma cell TC-ET after treatment with PCI-24781.
Since Pgp1 plays an important role in multidrug resistance of cancer cells, the expression of Pgp1 was evaluated in all multidrug resistant sarcoma cells and their parental cells, respectively. Western blot demonstrated that Pgp1 was over-expressed in multidrug resistant osteosarcoma cells U-2 OS MR and KH OS R2, and multidrug resistant chondrosarcoma cell CS-ZR. The expression of Pgp1 was evaluated in these multidrug resistant sarcoma cells after treatment with PCI-24781. Western blot demonstrated that PCI-24781 had no influence on the expression of Pgp1 in these multidrug resistant sarcoma cells.
The rationale for the development of HDACIs as anticancer drugs resides in their abilities to inhibit tumor cell growth, induce differentiation, and lower apoptotic threshold in transformed cells 10, 11, 34. We have shown that sub-lethal concentrations of PCI 24781 sensitized drug resistant sarcoma cells to the induction of cell death by several chemotherapy drugs including doxorubicin, ET-743, and Zalypsis. Doxorubicin is one of the most commonly used anticancer drugs for the treatment of patients with sarcoma. ET-743 (Yondelis®; Trabectedin) and Zalpsis (PM00104) are novel marine derived DNA-targeting anticancer agents. ET-743 has been approved by the European Medicines Agency (EMEA) for patients with advanced sarcomas who have either progressed after treatment with an anthracycline or are not clinically suitable to receive conventional agents. The current study demonstrates that PCI-24781 can induce apoptosis and inhibit the growth of multidrug resistant sarcoma cell lines. The accumulation of various biomarkers of apoptosis, including acetylated histones and p21, suggests PCI-24781 induces apoptosis.
Another property of HDAC inhibitors is their proposed ability to enhance the anticancer activity of numerous chemotherapeutic agents. Thus, the full potential of these drugs may be best realized in a combination of chemotherapy. Their ability to enhance the efficacy of many chemotherapeutic agents is likely due to a variety of anticancer effects. These effects include (1) increasing the acetylation of core histones, resulting in an open chromatin configuration that is more accessible to DNA-targeting agents,(2) shifting the balance of pro- and anti-apoptotic genes toward apoptosis, (3) inducing generation of reactive oxygen species (ROS), and (4) inhibiting angiogenesis, all of which contribute to their anticancer activity13, 35-37.
In the present study, we found PCI-24781 to have synergistic effects with chemotherapeutic drugs, including doxorubicin, Zalypsis, and ET-743 in multidrug resistant sarcoma cell lines. The MTT assay demonstrated that PCI-24781 reverses drug resistance in all four multidrug resistant sarcoma cell lines. Consistent with previous studies, we found Caspase 3 and 7, two caspases that are responsible for the majority of intracellular caspase-induced cleavages and cell death, are induced by PCI-24781 in multidrug resistant sarcoma cell lines. The caspase 3/7 activity assay demonstrated that PCI-24781 has a synergistic effect with other chemotherapeutic drugs to induce apoptosis in multidrug resistant sarcoma cell lines. In addition, the expression of RAD51 was found to be inhibited in all four sarcoma cell lines after treatment with PCI-24781. This indicates that inhibiting the expression of RAD51 by PCI-24781, which results in a decrease in homology-directed repair of DSBs, may play a role in the mechanisms underlying the anticancer synergy between PCI-24781 and other chemotherapeutic drugs in multidrug resistant sarcoma cell lines.
Furthermore, Gadd45α, which plays an important role in both cell cycle control, survival and apoptosis, was found to be induced in multidrug resistant sarcoma cell lines treated with PCI-24781, although cells treated with chemotherapy alone did not express GADD45α. Several chemotherapy drugs induced apoptosis in cancer cells by activating GADD45α. We found treatment of U-2 OS MR and KH OS R2 by PCI-24781 induced dose dependent Gadd45α increase in expression (Fig. 4). Our data suggests one potential mechanism of PCI-24781 reverses drug resistance was by activation of GAdd45α in some multidrug resistant osteosarcoma cell lines. This will in turn result in the down-regulaton of the threshold of apoptosis for osteosarcoma cells, undergoing apoptotic cell death triggered by other drugs. On the other hand, GADD45α was found not induced in multidrug resistant chondrosarcoma cell line CS-ZR and multidrug resistant Ewing’s sarcoma cell line TC-ET by PCI-24781, suggest GADD45α independent mechanisms may be exist in these PCI-24781 treated osteosarcoma cell lines. Our results provide evidence for the requirement of both the GADD45α-dependent and the GADD45α--independent apoptotic pathways in the PCI-24781-mediated death of drug-resistant sarcoma cells. We also found that Pgp1 expression in multidrug resistant cell lines were not affected by treatment with the combination of chemotherapy drug and PCI-24781. These results indicate that PCI-24781 does not reverse the drug resistance in sarcoma cell lines by targeting the Pgp1 expression.
In conclusion, the present study demonstrates that the HDAC inhibitor PCI-24781 contains anti-tumor activity in multi-drug resistant sarcoma cell lines. PCI-24781 has a synergistic effect on chemotherapeutic drug-induced apoptosis and can reverse drug resistance in MDR sarcoma cell lines.
This project was supported by a grant from the Gattegno and Wechsler funds. Dr. Duan is supported, in part, through a grant from Sarcoma Foundation of America, and a grant from the National Cancer Institute, NIH (Nanotechnology Platform Partnership), R01-CA119617. Dr. Choy is supported by the Jennifer Hunter Yates Sarcoma Foundation.