Date Published: July 10, 2017
Publisher: Public Library of Science
Author(s): Patrick Naumann, Jakob Liermann, Franco Fortunato, Thomas E. Schmid, Klaus-Josef Weber, Jürgen Debus, Stephanie E. Combs, Aamir Ahmad.
Sulforaphane (SFN), an herbal isothiocyanate enriched in cruciferous vegetables like broccoli and cauliflower, has gained popularity for its antitumor effects in cell lines such as pancreatic cancer. Antiproliferative as well as radiosensitizing properties were reported for head and neck cancer but little is known about its effects in pancreatic cancer cells in combination with irradiation (RT).
In four established pancreatic cancer cell lines we investigated clonogenic survival, analyzed cell cycle distribution and compared DNA damage via flow cytometry and western blot after treatment with SFN and RT.
Both SFN and RT show a strong and dose dependent survival reduction in clonogenic assays, an induction of a G2/M cell cycle arrest and an increase in γH2AX protein level indicating DNA damage. Effects were more pronounced in combined treatment and both cell cycle perturbation and DNA damage persisted for a longer period than after SFN or RT alone. Moreover, SFN induced a loss of DNA repair proteins Ku 70, Ku 80 and XRCC4.
Our results suggest that combination of SFN and RT exerts a more distinct DNA damage and growth inhibition than each treatment alone. SFN seems to be a viable option to improve treatment efficacy of chemoradiation with hopefully higher rates of secondary resectability after neoadjuvant treatment for pancreatic cancer.
Despite its low incidence pancreatic cancer is still the fourth leading cause of cancer death. While progress and innovation in oncology managed to improve 5-year survival rates of all tumor entities by approximately 20%, advances have been slow for pancreatic cancer . Main reasons are lack of early symptoms with subsequent late diagnosis mostly at advanced or even metastasized stages as well as a relative resistance to chemotherapeutics and irradiation (RT) . Hence, until today surgical resection is the only realistic chance for cancer cure. Unfortunately, at time of diagnosis less than one fourth of all patients have a disease that is amenable to surgical resection. Therefore, neoadjuvant treatment concepts are still in demand [3, 4]. In locally advanced stage, which comprises none metastasized but due to vessel involvement inoperable tumors, neoadjuvant chemoradiation is a reasonable treatment choice to potentially reach secondary resectability that improves survival rates significantly [3, 5, 6].
This study presents in vitro data showing enhanced antitumor efficacy in four pancreatic cancer cell lines by combining conventional photon irradiation with the phytochemical agent SFN. Treatment efficacy was assessed by clonogenic survival assays as well as flow cytometric tests for cell cycle perturbation and pan-nuclear γH2AX accumulation as surrogate for DNA double strand breaks. Both cell cycle blockade and DNA damage are cornerstones in radiation mediated antitumor effects. Since radiation induced growth inhibition mostly occurs in a longer time scale than cytotoxic chemotherapy, clonogenic assays still represent the traditional gold standard . In all our four established pancreatic cancer cell lines AsPC-1, BxPC-3, MIA PaCa-2 and Panc-1 we observed that treatment with SFN and/or exposition to irradiation leads to a dose dependent and highly significant reduction of clonogenic survival especially in combined treatment. SFN is known as potent inducer of apoptosis as well as macroautophagy in pancreatic cancer cells [15, 22]. Its effects in humans are currently investigated in a clinical phase I study (POUDER-trial) for patients with metastasized pancreatic cancer . Efficacy of RT related cytotoxicity in pancreatic cancer can be enhanced by combination with chemotherapeutics such as gemcitabine [24, 25]. As a matter of fact, chemoradiation as neoadjuvant treatment concept is–if in concern of patient’s general condition reasonable administrable—still more a treatment of choice than RT alone [4, 26]. Combination of SFN and subsequent irradiation in head and neck cancer cell lines inhibited cell proliferation more than each treatment alone . In murine osteosarcoma cells combination of SFN and irradiation has been described to be superior to single treatments concerning growth inhibition, cell cycle perturbation and apoptosis induction .
In conclusion, SFN and RT cause growth inhibition by cell cycle perturbation and DNA damage. Combined treatment had more pronounced effects and lasted over longer periods than each treatment alone. Our data provide good in vitro evidence to infer that co-treatment with SFN may enhance effects of RT in pancreatic cancer.