Synergistic Antitumor Interactions Between Gemcitabine And Chidamide Or Triptolide In Pancreatic Cancer Celts

Pancreatic adenocarcinoma is a high incidence and mortality gastrointestinalmalignancy worldwidely. Approximately200,000victims die from the disease eachyear in the world, and the incidence and mortality are rapidly increasing in recentyears. Pancreatic cancer has a very poor prognosis due to its aggressiveness with a5-year survival rate less than5%. Surgical resection provides the only potential curefor pancreatic cancer, yet only10~20%patients are suitable for surgeries while in theother80%showing metastasis was found at the first time of diagnosis of pancreaticcancer. Adjuvant therapy, especially chemotherapy, is the main treatment forpost-surgical pancreatic cancer patients and advanced pancreatic cancer patients.Gemcitabine (2’,2’-difluorodeoxycytidine, dFdC) is the first-line chemotherapyfor the treatment of pancreatic cancer approved by US FDA, and provides modestsurvival benefit in patients. The phase III clinical trial of dFdC showed the mediansurvival time of patients with advanced pancreatic cancer on dFdC was5.6monthswhile those on5-FU was4.4months. However, even with this drug, most pancreaticadenocarcinomas show a high degree of inherent and acquired chemoresistance togemcitabine.Histone acetylation, one of the epigenetic regulations, is a posttranslationalmodification of the nucleosomal histones that affects chromatin structure andmodulates gene expression. The acetylation status of histones is regulated by twoopposing family of enzymes: histone acetyltransferases (HATs) and histonedeacetylases (HDACs). HDACs are found to be overexpressed in many types oftumors and are associated with tumorigenesis. Thus, targeting HDACs represents apromising strategy for cancer therapy. Previous and ongoing studies havedemonstrated that HDAC inhibitors (HDACIs) are promising new agents for thetreatment of cancers and have shown anticancer activities over a wide spectrum of malignancies, most impressively in hematological cancers. Chidamide(CS055/HBI-8000), a novel HDACI of the benzamide class, has been in phase Iclinical trial in the United States of America and phase II/III trials for cutaneous T-celllymphoma (CTCL) and peripheral T-cell lymphoma (PTCL) in China. Chidamide hasshown anticancer activity in colon, lung, breast, and liver solid tumor cells and inmyeloid leukemia cells.Triptolide, a diterpene triepoxide, is a major active component of extractsderived from the medicinal plant Tripterygium wilfordii Hook F (TWHF). Triptolidehas multiple pharmacological activities and has been widely used to treatinflammatory and autoimmune diseases. It has also been used in organ transplantation.In addition to the anti-inflammatory and immunosuppressive activities, triptolide alsoexhibits a strong anti-proliferative and anticancer effect and can sensitize solid tumorcells to chemotherapeutic agents.In this study, we sought to determine whether chidamide inhibits proliferation ofpancreatic cancer cells and whether chidamide or triptolide can synergisticallyenhance the anticancer activity of gemcitabine. We have demonstrated that chidamideinduces growth inhibition of pancreatic cancer cells. Further, chidamide or triptolidealso significantly enhances the apoptosis-inducing effects of gemcitabine inpancreatic cancer cells. This study comprises of the following three parts.1. Chidamide inhibits the proliferation of pancreatic cancer cellsChidamide treatments of the pancreatic cancer cell lines BxPC-3or PANC-1resulted in growth inhibition in a dose-and time-dependent manner. This wasaccompanied by increased levels of acetylated histone H3in both BxPC-3andPANC-1cells. These results indicated that chidamide has potential anticancer activityby effectively inhibiting HDAC enzymatic activities in pancreatic cancer cells.Chidamide treatments also induced G2/M cell cycle arrest in a dose-dependentfashion in the the pancreatic cancer cell lines, along with increased levels of p21.Further, chidamide treatments of BxPC-3and PANC-1cells resulted indose-dependent induction of reactive oxygen species (ROS), which resulted in DNA damage.2. Chidamide and gemcitabine synergistically inhibited proliferation and inducedapoptosis of pancreatic cancer cellsWhen administered sequentially (gemcitabine first followed by chidamide),chidamide synergistically enhanced gemcitabine-induced growth inhibition of thepancreatic cancer cells, reflected by the decreased gemcitabine IC50s (2.6-to9.2-foldin BxPC-3cells and1.8-to7.7-fold in PANC-1cells, respectively). Further,treatments of the pancreatic cancer cell lines with chidamide significantly enhancedgemcitabine-induced apoptosis determined by flow cytometry analysis with annexin vand propidium iodide standing and Western blotting measuring cleavage of caspase3and caspase9. Treatments of the BxPC-3and PANC-1cells with combinedgemcitabine and chidamide resulted in cooperative decrease of mitochondriamembrane potential (MMP). This was accompanied by decreased expression of theanti-apoptotic Bcl-2family protein Mcl-1, increased expression of the proapoptoticBcl-2family proteins Bax and Bim, and cleavage (activation) of the proapoptoticBcl-2famaily protein Bid. These results demonstrate that treatments of pancreaticcancer cells with combined gemcitabine and chidamide promote membranepermeabilization of mitochondria to trigger apoptosis. Chidamide also enhanced Sphase arrest induced by gemcitabine accompanied by increased DNA damage. Thismay potentially due to suppression of the expression of DNA repair genes Chk1andBRCA1by chidamide in the cells.3. Triptolide and gemcitabine synergistically inhibited proliferation and inducedapoptosis of pancreatic cancer cellsAnalogous to the combination of gemcitabine and chidamide, when administeredsequentially, triptolide significantly reduced gemcitabine IC50s in both BxPC-3andPANC-1cells. The extent and direction of antitumor interactions between the twoagents were obviously synergitic when determined by the CalcuSyn software analyses.In addition, triptolide significantly augmented gemcitabine-induced apoptosis in thepancreatic cancer cells determined by flow cytometry analyses and Western blotsmeasuring cleavage of caspases3and9. Treatments of the pancreatic cancer cells with combined triptolide and gemcitabine also resulted in cooperative decrease ofMMP, analogous to that of the combination of gemcitabine and chidamide. This wasaccompanied by increased expression of Bax and decreased expression of Bcl-2andMcl-1. Interestingly, triptolide enhanced gemcitabine-induced S phase arrest andDNA damage in the pancreatic cancer cells, as well. Furthermore, triptolidesuppressed the expression of Chk1and of the PI3K/Akt pathway.In summary, chidamide or triptolide synergistically enhances gemcitabinecytotoxicity against pancreatic cancer cells in vitro. However, these in vitro findingsneed follow up studies in vivo. Nonetheless, our data suggest that cotreatment ofpancreatic cancer patients with gemcitabine and chidamide or triptolide may provideadditional clinical benefits.