- Top of page
- Materials and Methods
- Disclosure Statement
Histone deacetylase inhibitors are a group of recently developed compounds that modulate cell growth and survival. We evaluated the effects of the histone deacetylase inhibitor MGCD0103 on growth of pancreatic carcinoma models following single agent treatment and in combination with gemcitabine. MGCD0103 inhibited tumor cell growth and acted synergistically with gemcitabine to enhance its cytotoxic effects. Gene expression analysis identified the cell cycle pathway as one of the most highly modulated gene groups. Our data suggest that MGCD0103 + gemcitabine might be an effective treatment for gemcitabine-refractory pancreatic cancer. (Cancer Sci 2011; 102: 1201–1207)
Pancreatic cancer (PC) is a devastating and universally fatal disease, with a 5-year survival rate of 5%. Despite recent efforts to improve early diagnosis, staging, and surgical and chemotherapeutic management of PC, survival remains poor.(1) This is due to the non-detectable development of tumors, aggressive phenotype, rapid and severe patient debilitation, and radio- and chemoresistance to many drugs. Although systemic chemotherapy with gemcitabine has led to modest clinical benefits in patients, the combined use of other cytotoxics with gemcitabine has failed to improve survival.(2) This underlies the need to explore new targets and molecular events in order to treat PC patients in a multimodal and multitargeted manner.
Histones are small proteins that complex with DNA to form the nucleosome core and chromatin that package the human genome.(3) Histone acetylation is a posttranslational modification that allows for “relaxation” of chromatin structure, subsequent accessibility of transcription factors to DNA and transcriptional activation. Deacetylation of histone tails by histone deacetylase (HDAC) induces transcriptional inactivation through chromatin condensation.(4) There are 18 known human HDACs classified into four groups based on phylogenetic and functional criteria. MGCD0103 inhibits the class I HDACs (1–3 and 8) that typically associate with multiprotein repressor complexes and are thought to play a role in cell survival and proliferation.(5)
Alterations in HDACs are found in many human cancers including pancreatic adenocarcinoma.(5) Abnormally high levels of HDAC1 in up to 40% of cancer cells and their surrounding “normal” tissues was observed(6) and high HDAC1 expression correlates with advanced stage lung and pancreatic cancer.(7,8) In many tumor types including PC, aberrant transcriptional silencing of tumor suppressor genes contributes to transformation and malignant growth. Reactivation of these epigenetically silenced genes by HDAC inhibitors (HDACi) has resulted in antitumor effects such as apoptosis, cell cycle arrest, differentiation, and senescence.(9) Histone deacetylases have also been shown to induce re-expression of a number of proteins such as the cell cycle inhibitor p21, estrogen receptor, and pro-apoptosis proteins such as caspase-3, PARP, and BAX.(5)
Conventional chemotherapeutic drugs target both normal and tumor cells, but HDACi appear to be cytotoxic to malignant cells, sparing the normal cells.(10) Because of their diverse antitumor effects and largely manageable side-effects, HDACi are candidates for use in combination with cytotoxics, targeted therapy, or other epigenetic modifiers. For example, entinostat (MS-275) and vorinostat (SAHA) have been shown to chemosensitize breast and pancreatic tumor cells to adriamycin and gemcitabine, respectively.(9,11,12) Currently, there are two HDACi that are clinically approved: vorinostat and romidepsin for the treatment of cutaneous T-cell lymphoma.
MGCD0103, an isotype-selective HDACi, has been clinically evaluated for the treatment of hematologic malignancies and advanced solid tumors, alone and in combination with standard-of-care agents. MGCD0103, a compound with favorable pharmacokinetic/pharmacodynamic profiles, showed mechanism-based antileukemia activity in a recent phase I trial and was also deemed tolerable in a subsequent advanced solid tumor trial.(13,14) MGCD0103 was evaluated for activity in combination with gemcitabine in refractory solid malignancies and in locally advanced or metastatic pancreatic adenocarcinoma. Findings from this clinical study suggest that the combination might have clinical activity in patients with refractory pancreatic cancer.(15) To better understand the potential mechanism of MGCD0103 in pancreatic cancer, we evaluated the ability of MGCD0103 to synergize with gemcitabine in pancreatic tumor models.
- Top of page
- Materials and Methods
- Disclosure Statement
MGCD0103 is an orally available, isotype-specific HDACi that is being evaluated as a single agent and in combination with chemotherapy for treatment of both hematologic and solid malignancies. In vitro, MGCD0103 has potent antiproliferative activity against human tumor cell lines but not against normal cells at submicromolar to micromolar concentrations.(22–25) In pharmacological models, MGCD0103 showed significant antitumor activity at well-tolerated doses in a variety of tumor types including colon, non-small-cell lung, pancreatic, and prostate cancer xenografts. Notably, drug was detected in the plasma up to 24 h post-dose and for the first 8 h, plasma concentrations were at or above the EC50 values needed to inhibit HDACs 1 and 2 in vitro.(22,24)
MGCD0103 has also been evaluated in several clinical trials. Pharmacokinetic profile was favorable with dose-dependent exposure and a half-life of approximately 10 h regardless of dosing schedule.(13,14,23,26) MGCD0103 as a single agent displays antitumor activity in hematological malignancies as well as in lymphoproliferative disease.(13,27,28) However, single agent effects in solid tumor patients have been less straightforward. In a phase I study of patients with advanced solid tumors who were treated with up to 45 mg/m2 (equivalent to a fixed dose of 90 mg) MGCD0103 three times per week, a Cmax of approximately 172 ng/mL (0.43 μM) was reached. Drug was tolerated and the most frequently observed side-effects were fatigue and gastrointestinal -related symptoms. Stable disease, but no objective tumor response, was observed. In a second, as yet unpublished phase I/II clinical study, patients with refractory and/or advanced solid tumors were treated with ascending doses (fixed dose of 50–110 mg; 90 mg was the maximum tolerated dose and recommended dose) of MGCD0103 in combination with gemcitabine. Dose-limiting toxicities included fatigue, diarrhea, vomiting, and nausea; all were expected with such a combination treatment. Interestingly, a study update reported two partial responses out of five pancreatic carcinoma patients and two additional partial responses in patients with nasopharygeal carcinoma and cutaneous T-cell lymphoma. Although this is only preliminary data from a very small number of patients, it is promising and suggests that solid tumor efficacy may be reached by combining MGCD0103 with gemcitabine.(15)
In the current study, we evaluated the in vitro effects of the HDACi, MGCD0103, on human pancreatic carcinoma cell lines and explant growth. Micromolar concentrations of MGCD0103 inhibited cell proliferation in a dose-dependent manner, both on plastic as well as in a 3-D clonogenic assay and the monolayer EC50 values are consistent with those determined in previous in vitro studies.(22,24,25) However, most of the pancreatic cell lines and explants were not completely inhibited by MGCD0103 at concentrations comparable to levels attainable in clinical dosing regimens. Therefore, we next evaluated a dose range of MGCD0103 (including clinically achievable, submicromolar concentrations) in combination with gemcitabine in the tumor cell lines at three different treatment schedules (concurrent treatment, MGCD0103 pretreatment, and gemcitabine pretreatment) and found that, in all cases, MGCD0103 synergized with gemcitabine to induce cytotoxicity. Our drug combination data indicates that clinically attainable concentrations of MGCD0103 could be effective in synergizing with gemcitabine and that sensitization by MGCD0103 may be independent of treatment sequence, potentially allowing for more flexible dosing schedules in the clinic.
Resistance to the cytotoxic effect of gemcitabine could be associated with multiple mechanisms including alteration of apoptosis genes, changes in drug transport and cellular turnover, as well as decreased expression or sensitivity of drug targets.(29–32) If agents such as HDACi can enhance gemcitabine-associated cytotoxicity pathways or activate novel pathways leading to cell death, gemcitabine refractory tumors could be rendered responsive to treatment. It has been previously shown that SAHA and trichostatin A, two HDACi, induce the upregulation of cyclin-dependent kinase inhibitor p21,(33,34) facilitating apoptosis of pancreatic carcinoma cell lines. Both of these inhibitors also potentiate the effects of gemcitabine on growth inhibition. MGCD0103 has been shown to induce cell cycle arrest (G1 and G2/M) in various human cancer cell lines(22) and in the current study, we propose that one of the mechanisms by which MGCD0103 could induce pancreatic carcinoma cell growth arrest is through upregulation of p21 expression. Unlike gemcitabine, which primarily leads cells into G1 arrest, p21 is thought to promote arrest in both the G1 and G2/M phase of the cell cycle.(35–38) Therefore, as we observed in our experiments, treatment of tumor cells with an HDACi such as MGCD0103 is expected to enhance growth inhibition compared to treatment with gemcitabine alone. To further elucidate potential mechanisms of synergy between MGCD0103 and gemcitabine, additional studies should focus on evaluating apoptotic/autophagic proteins, markers of antitumor immunity, and cell differentiation in MGCD0103-treated pancreatic cells to identify unique pathways that are stimulated by MGCD0103 and which might lead to synergy with gemcitabine. Indeed, preliminary data showed that combined treatment with MGCD0103 plus gemcitabine in the Panc-1 pancreatic cancer cell line synergistically induced apoptosis (Nguyen H., Li Z., Martell R. E., Besterman J. M., 2005, unpublished data).
Chemotherapeutic treatment options for advanced and metastatic pancreatic cancer are currently limited and an efficient and well-tolerated option is urgently needed. Most of the single chemotherapy agents used to treat pancreatic cancer yield low response rates and only a minimal impact on survival. Although gemcitabine is currently the standard-of-care for advanced disease, it appears to improve the quality of life for a short time only and clinical efficacy is poor. Our study showed that MGCD0103 has inhibitory effects on pancreatic cancer cell growth both as a single agent and in combination with the standard-of-care gemcitabine, and that cell cycle inhibition is one potential pathway through which MGCD0103 acts. Although additional studies are necessary in order to better understand its multiple mechanisms of action, these data suggest that MGCD0103 may be an effective treatment for pancreatic cancer patients, even those who have previously been shown to be refractory to gemcitabine.