PDAC remains a therapeutic challenge with a poor overall prognosis. Only surgery with adjuvant chemotherapy can achieve a long-term perspective in patients with localized tumours [13
]. Adjuvant postoperative chemotherapy based on 5-FU or gemcitabine has been shown to improve the survival of these patients [16
]. However, even under optimal treatment conditions, the 5-year survival rate doesn’t exceed 25% [13
]. Additionally, palliative treatment in advanced tumour stages is associated with a poor prognosis and a median survival of about 6
months. To improve this situation, investigation of new therapeutic agents for PDAC treatment is essential.
The family of HDACi represents a novel approach in oncological research. In defined - predominantly haematological - tumour entities, HDACi have already passed the stage of experimental research and been investigated clinically [17
]. Regarding PDAC, promising results have been shown using SAHA, TSA, butyrate and some other histone deacetylase inhibitors in experimental studies [6
]. Belinostat is a novel member of the family with a distinct pan-HDAC inhibitory effect. It has been shown to be strongly effective in experimental settings of ovarian, bladder and colon cancer, as well as haematological tumour entities [9
]. Consecutive clinical trials have proven an anti-tumour effect of belinostat as a monotherapy in T-cell lymphomas and thymomas. In addition, belinostat has demonstrated beneficial effects in combination with other anti-cancer drugs for the treatment of ovarian and bladder cancer, CUP, multiple myeloma and acute myeloid leukaemia. Despite these findings, no data are available concerning belinostat in the context of PDAC treatment. Consequently, in the present study, the efficacy of belinostat for PDAC treatment was investigated in experimental in vitro
and in vivo
settings for the first time.
Comparable to the results of previous studies in bladder [8
], colorectal [25
] or hepatocellular carcinoma [21
], we found a strong dose dependent antiproliferative activity of belinostat in three pancreatic cancer cell lines (T3M4, Panc-1 and AsPC-1) with an IC50 concentration in the nanomolar range, similar to other tumour entities [8
This antiproliferative effect can be explained by a strong proapoptotic activity in pancreatic cancer cells, demonstrated by annexinV/propidium iodide staining. This is in line with other studies on AML- [26
] and hepatocellular carcinoma cells [21
], underlining that apoptosis induction is an important mechanism of the anti-tumourous effect of HDACi, and particularly belinostat. As apoptosis induction is an important mechanism of anti-cancer chemotherapy [27
], we tested the influence of concomitant use of belinostat and gemcitabine. As described in studies with other HDACi like trichostatin A [6
] and 4-phenylbutyrate [18
], the combination of gemcitabine and belinostat strongly enhanced the proapoptotic effects of each substance alone. This may be due to the expression of proapoptotic proteins like Caspase-8 and Bid, and activation of the gemcitabine-mediated JNK-pathway [18
Increase in histone H4 acetylation has been shown to be helpful in monitoring belinostat activity [28
]. Consequently, we examined belinostat-dependent expression of acH4 in PDAC cells. Acetylation of H4 was increased in all cell lines tested, confirming the inhibitory effect of belinostat on HDAC activity in pancreatic cancer cells.
Cyclin-dependent kinase inhibitor p21Cip1/Waf1
is a key protein participating in cell cycle regulation. Previous studies have shown that HDACi activates expression of p21Cip1/Waf1
through enhanced histone acetylation around the p21Cip1/Waf1
]. We performed western blot analysis with treated and control Panc-1 cells to clarify the effect of belinostat on p21Cip1/Waf1
expression. Belinostat induced an upregulation of p21Cip1/Waf1
, as has been described for other HDAC inhibitors in pancreatic cancer [18
]. Increased expression of p21Cip1/Waf1
in these studies was associated with normalization of the cell cycle and induction of apoptosis.
Regarding the effect of belinostat in vivo
, we observed that belinostat was an effective growth inhibitor of T3M4 pancreatic cancer cells in a nude mouse model. Mice treated with belinostat showed xenograft growth inhibition for more than 28
days after tumour inoculation, without any signs of toxicity. The reduction in the tumour volume was associated with decreased cell proliferation, as shown by Ki-67 immunohistochemistry. Similar observations in in vivo
tumour models were shown in previous studies, e.g. in human ovarian cancer s.c. xenografts; the efficacy of the treatment with belinostat was further enhanced when a combination therapy with carboplatin was added [24
]. Plumb et al. [22
] described a significant dose-dependent growth delay of human colon tumour xenografts in mice after belinostat treatment, without signs of toxicity.
In contrast to our in vitro observations, we could not find an additional effect of combined therapy with belinostat and gemcitabine in vivo. A possible explanation for this discrepancy is the relatively high dosage of gemcitabine administered in the in vivo study. This might have covered a possible additional belinostat effect.