_:b36443790 "discussion" . . _:b567469109 "2"^^ . _:b567469090 . _:b567469079 . _:b36443781 "Viable and dead cells were identified by their ability and inability to exclude vital dyes, respectively [>>48<<]. In brief, cells were stained with 0.2% trypan blue, and the numbers of viable and dead cells were determined using a hemocytometer. Cell viability (%) was defined as 100 \u00D7 \u2018the number of viable cells\u2019/ (\u2018the number of viable cells\u2019 +" . . _:b567469108 "2"^^ . . _:b567469096 . _:b36443807 "Unlike in normal pancreas, JNK is activated in the majority of pancreatic cancers [>>42<<]. The significance of JNK activation in pancreatic cancer biology remained unclear, but we have only recently demonstrated that JNK activation by KRas, which is mutationally activated in the majority of pancreatic cancers, plays a key" . _:b36443800 "It has been demonstrated that CSCs have lower intracellular ROS levels and that the low ROS levels are associated with the radioresistance of CSCs [31, >>32<<]. In contrast to radioresistance, however, the role of enhanced ROS defense of CSCs in chemoresistance has yet to be established. Significantly, lines of evidence suggest that ROS may participate in the cytotoxic mechanisms of" . _:b36443798 . _:b567469105 "2"^^ . . _:b567469104 "2"^^ . . _:b36443811 "On the other hand, GEM for instance reportedly exerts its cytotoxic effect on lung cancer cells through activation of JNK [>>45<<], suggesting the possibility that co-treatment could render the cells even more resistant to chemotherapeutic agents in case their cytotoxic effect is JNK-dependent." . _:b567469107 "2"^^ . . _:b567469106 "2"^^ . . . _:b36443797 . _:b36443803 "In sharp contrast to the well-documented role of ROS in JNK activation [>>38<<, 39], the role of JNK in the control of the intracellular ROS level remains largely uninvestigated." . _:b567469026 . _:b36443778 . _:b36443803 . . . _:b567469052 . _:b36443777 . _:b36443776 . _:b567469106 . . . . _:b36443806 . _:b36443787 . _:b567469055 . _:b36443786 . _:b36443791 . . . . _:b36443785 . _:b567468989 "5"^^ . _:b567468988 "5"^^ . . _:b567468991 "4"^^ . _:b567469016 . _:b567468990 "5"^^ . _:b567468986 "6"^^ . . _:b567469088 . . _:b567468987 "6"^^ . _:b567469049 . _:b567469066 . _:b567469056 . _:b567469031 . _:b567469032 . _:b567468985 "8"^^ . _:b36443781 . _:b567468997 "3"^^ . _:b567468996 "3"^^ . _:b567469060 . _:b567468999 "3"^^ . _:b36443788 . . _:b567468998 "3"^^ . _:b567469001 . _:b567468993 "4"^^ . . _:b567469089 . . _:b567469007 . _:b567468992 "4"^^ . . _:b567469082 . . _:b567468995 "4"^^ . . . _:b567468994 "4"^^ . _:b36443791 . . _:b567469005 "3"^^ . . _:b567469004 "3"^^ . _:b567469007 "3"^^ . . _:b567469099 . _:b567469006 "3"^^ . _:b567469001 "3"^^ . _:b36443768 "Pancreatic cancer is among the most intractable of all human cancers and is also a leading cause of cancer-related death in the world [>>1<<, 2]. Besides the difficulty of early detection, which leads to dismally low surgical resection rate, pancreatic cancer is characterized by pervasive therapy resistance, being refractory to both chemotherapy and radiotherapy [3\u20135]." . _:b36443779 . _:b567469000 "3"^^ . _:b567469003 "3"^^ . _:b567469002 "3"^^ . . _:b36443773 . _:b567469013 "3"^^ . _:b567468989 . _:b567469012 "3"^^ . _:b567468988 . _:b567469015 "2"^^ . _:b567469097 . _:b567468991 . _:b567469014 "3"^^ . _:b567468990 . _:b567468992 . _:b36443802 . _:b567469009 "3"^^ . _:b567469058 . _:b36443771 . _:b567468985 . _:b567469008 "3"^^ . _:b567469050 . _:b567469011 "3"^^ . . _:b36443794 . _:b567468987 . _:b36443784 "CSCs are in general considered to be highly resistant to chemotherapeutic drugs [9, >>10<<]. To examine whether this applies to the pancreatic CSCs we have established, we used PANC-1 CSLCs, CSCs derived from the pancreatic cancer cell line PANC-1 [15], and determined the sensitivity of self-renewing (undifferentiated) PANC-1" . _:b567469010 "3"^^ . _:b567468986 . _:b567469068 . _:b567469021 "2"^^ . . _:b567469059 . . _:b567468997 . _:b567469020 "2"^^ . . _:b567468996 . _:b567469023 "2"^^ . _:b567468999 . . _:b567469022 "2"^^ . _:b567469033 . _:b36443807 . _:b567468998 . _:b567469017 "2"^^ . _:b567468993 . . _:b567469016 "2"^^ . _:b567468992 . _:b567469019 "2"^^ . _:b567468995 . _:b567469018 "2"^^ . . . _:b567468994 . _:b567469029 "2"^^ . _:b567469005 . _:b567469028 "2"^^ . _:b567469084 . _:b567469057 . _:b567469011 . _:b567469004 . _:b567469031 "2"^^ . _:b567469108 . _:b567469100 . _:b567469007 . _:b567469030 "2"^^ . . _:b567469006 . _:b36443769 "Pancreatic cancer is among the most intractable of all human cancers and is also a leading cause of cancer-related death in the world [1, >>2<<]. Besides the difficulty of early detection, which leads to dismally low surgical resection rate, pancreatic cancer is characterized by pervasive therapy resistance, being refractory to both chemotherapy and radiotherapy [3\u20135]." . _:b567469092 . _:b567469025 "2"^^ . _:b36443809 . _:b567469001 . _:b567469024 "2"^^ . _:b567469000 . _:b567469027 "2"^^ . "PMC0" . . _:b567469003 . _:b567469026 "2"^^ . _:b567469002 . _:b567469036 . _:b567469037 "2"^^ . _:b567469013 . _:b567469036 "2"^^ . _:b567469034 . _:b567469012 . _:b567469039 "2"^^ . . _:b567469015 . _:b36443796 "Indeed, there are studies suggesting that the JNK pathway may play a positive role in chemoresistance of some cancer types [>>26<<]. However, the role of JNK in chemoresistance/sensitivity of pancreatic cancer cells, let alone pancreatic CSCs, to clinically relevant chemotherapeutic agents has remained largely unknown except for a report showing that GEM-induced" . _:b567469038 "2"^^ . _:b567469014 . _:b567469033 "2"^^ . _:b567469009 . _:b567469032 "2"^^ . _:b36443779 "Normal human IMR90 fetal lung fibroblasts [>>46<<, 47] were obtained from American Type Culture Collection and maintained in DMEM/F12 supplemented with 10% fetal bovine serum (FBS) and 100 units/mL penicillin and 100 \u03BCg/mL streptomycin." . _:b567469008 . _:b567469035 "2"^^ . . _:b567469011 . _:b567469034 "2"^^ . _:b567468998 . _:b36443774 "CSCs in the poor prognosis of pancreatic cancer, suggesting that overcoming the chemoresistance inherently associated with pancreatic CSCs is apparently an indispensible step to improve overall management of pancreatic cancer [13, >>14<<]. However, to date, the molecular basis on which pancreatic CSCs acquire resistance to conventional chemotherapeutic agents against pancreatic cancer has been poorly understood." . _:b567469010 . _:b567469045 "2"^^ . . . _:b567469021 . _:b567469044 "2"^^ . _:b567469020 . _:b567469047 "2"^^ . _:b567469023 . _:b567469046 "2"^^ . . _:b36443793 . _:b567469022 . _:b567469041 "2"^^ . _:b36443808 "we have only recently demonstrated that JNK activation by KRas, which is mutationally activated in the majority of pancreatic cancers, plays a key role in the maintenance of self-renewal and tumor-initiating capacity of pancreatic CSCs [>>15<<]. The findings of the present study now add a new role of JNK in pancreatic CSCs, namely, the maintenance of chemoresistance." . _:b567469017 . _:b567469040 "2"^^ . _:b36443782 "results" . . . _:b36443810 "S, and M.O., unpublished observation), suggesting that \u201Crewiring\u201D of signaling pathways [>>44<<] may have taken place with some stability during the pre-treatment period, i.e., by the time cells were treated with 5-FU." . _:b567469016 . _:b567469043 "2"^^ . _:b567469019 . _:b567468994 . _:b567469042 "2"^^ . _:b567469085 . . _:b567469018 . _:b567469053 "2"^^ . _:b36443797 "let alone pancreatic CSCs, to clinically relevant chemotherapeutic agents has remained largely unknown except for a report showing that GEM-induced apoptosis of pancreatic cancer cells was dependent on JNK-mediated DUSP1 expression [>>27<<]. In this regard, the present study is the very first to demonstrate that JNK has a protective role against 5-FU or GEM in pancreatic cancer cells, more specifically, in pancreatic CSCs." . _:b567469029 . _:b567469052 "2"^^ . _:b567469028 . _:b567469055 "2"^^ . _:b567469031 . _:b567469054 "2"^^ . _:b567469030 . _:b567469049 "2"^^ . . _:b567469025 . _:b567469048 "2"^^ . _:b567469024 . _:b567469051 "2"^^ . _:b567469087 . . _:b567469027 . _:b36443800 . _:b567469050 "2"^^ . _:b567469026 . _:b567469037 . _:b36443789 . _:b36443799 . _:b36443791 "Pancreatic cancer is one of the most malignant human cancers, which is so often unresectable already at the time of diagnosis and, above all, responds poorly to either chemotherapy or radiotherapy [>>3<<\u20135]. A growing body of evidence has come to suggest that pancreatic CSCs, endowed with both tumor-initiating capacity as well as with high therapy resistance, is among the key factors underlying the extreme refractoriness of pancreatic" . _:b567469036 . . . _:b567469039 . _:b36443802 "Significantly, lines of evidence suggest that ROS may participate in the cytotoxic mechanisms of chemotherapeutic agents such as 5-FU and GEM [17, >>33<<\u201337], lending support to the idea that cellular antioxidant capacity may determine cells' sensitivity/resistance to such chemotherapeutic agents." . _:b567469038 . _:b36443804 "In sharp contrast to the well-documented role of ROS in JNK activation [38, >>39<<], the role of JNK in the control of the intracellular ROS level remains largely uninvestigated." . _:b567469047 . . _:b36443796 . . _:b567469033 . _:b567469109 . . _:b36443805 . _:b567469032 . _:b36443780 . . _:b567469091 . _:b567469035 . . _:b567469034 . _:b36443798 . _:b567468995 . _:b567469045 . _:b567468989 . _:b567469044 . _:b36443793 "Here in this study, we first found that pancreatic CSCs, which we have shown to have activated JNK [15], were highly resistant to 5-FU and GEM compared with their non-stem counterparts, essentially consistent with earlier reports [>>13<<, 14]. We then demonstrated that inhibition of the activated JNK in pancreatic CSCs sensitized them to 5-FU and GEM and that JNK inhibition did so through elevation of the intracellular ROS level in combination with these chemotherapeutic" . _:b567468988 . _:b36443771 "improved with the development of fluorouracil (FU)- and gemcitabine (GEM)-based chemotherapeutic regimens, however, the prognosis of pancreatic cancer still remains miserably poor with only less than 10% of patients surviving 5 years [>>5<<\u20137]." . _:b567469047 . _:b567469046 . _:b567469062 . _:b567469041 . _:b567469040 . _:b567469043 . _:b567469042 . _:b567469061 . _:b567469053 . _:b567469052 . _:b36443774 . _:b567469051 . _:b36443772 "been associated with higher therapy resistance compared with non-stem cancer cells and are therefore increasingly deemed a major culprit of post-treatment progression and/or recurrence of a variety of, albeit not all, human cancers [>>8<<\u201312]. Since the identification of pancreatic CSCs, studies have consistently shown that pancreatic CSCs do have higher chemoresistance than non-stem pancreatic cancer cells and have also implicated pancreatic CSCs in the poor prognosis of" . . _:b567469055 . _:b567469014 . _:b567469018 . _:b567469054 . . . . _:b567469049 . _:b567469048 . _:b567469101 . _:b567469051 . _:b567469050 . . . . . . _:b36443770 . _:b567469037 . _:b567469042 . . . . _:b36443767 . _:b36443767 . . . . . . _:b567469020 . . . _:b36443775 . _:b567469107 . _:b567469072 . _:b36443801 "Significantly, lines of evidence suggest that ROS may participate in the cytotoxic mechanisms of chemotherapeutic agents such as 5-FU and GEM [>>17<<, 33\u201337], lending support to the idea that cellular antioxidant capacity may determine cells' sensitivity/resistance to such chemotherapeutic agents." . _:b36443798 . . _:b36443772 . _:b36443773 . _:b36443774 . _:b36443795 . _:b36443776 "The establishment of PANC-1 CSLCs and PSN-1 CSLCs, CSCs derived from human pancreatic cancer cell lines PANC-1 and PSN-1, respectively, was previously reported [>>15<<]. The authenticity of PANC-1 CSLCs and PSN-1 CSLCs as cells derived from PANC-1 and PSN-1, respectively, was verified by genotyping of short tandem repeat (STR) loci (Bio-Synthesis, Inc., Lewisville, TX, USA) followed by comparison to the" . . _:b36443775 . _:b36443768 . _:b567469040 . _:b567469021 . . _:b36443769 . _:b36443770 . _:b36443802 . . _:b36443771 . _:b36443770 "Besides the difficulty of early detection, which leads to dismally low surgical resection rate, pancreatic cancer is characterized by pervasive therapy resistance, being refractory to both chemotherapy and radiotherapy [>>3<<\u20135]. In the past decades, the survival of patients with pancreatic cancer has been improved with the development of fluorouracil (FU)- and gemcitabine (GEM)-based chemotherapeutic regimens, however, the prognosis of pancreatic cancer still" . _:b567468988 . _:b567468989 . _:b36443780 . _:b567468990 . _:b567468991 . _:b567468985 . _:b36443781 . . _:b567468986 . _:b567468987 . . _:b567469030 . . _:b36443782 . _:b36443783 . . _:b36443776 . _:b567469076 . . _:b36443777 . . _:b36443778 . . . . _:b36443779 . . . . _:b36443788 . . _:b36443789 . . _:b567469028 . _:b567469029 . _:b567469030 . _:b36443790 . _:b567469031 . . . _:b567469024 . _:b567469025 . _:b36443791 . _:b567469026 . _:b567469027 . _:b567469036 . _:b567469037 . _:b36443784 . _:b567469038 . _:b567469039 . _:b567469032 . _:b567469033 . _:b36443785 . _:b567469034 . _:b567469035 . _:b567469044 . _:b567469045 . _:b36443786 . _:b567469046 . _:b567469044 . . _:b567469047 . _:b567469040 . _:b567469041 . _:b36443787 . _:b567469042 . _:b567469043 . _:b567469052 . . _:b567469053 . _:b36443796 . _:b567469054 . _:b567469055 . _:b567469048 . _:b567469049 . _:b36443797 . _:b567469050 . _:b567469051 . _:b36443783 "CSCs are in general considered to be highly resistant to chemotherapeutic drugs [>>9<<, 10]. To examine whether this applies to the pancreatic CSCs we have established, we used PANC-1 CSLCs, CSCs derived from the pancreatic cancer cell line PANC-1 [15], and determined the sensitivity of self-renewing (undifferentiated)" . _:b567468996 . . _:b567468997 . _:b36443798 . _:b567468998 . _:b567468999 . _:b567468992 . _:b567468993 . _:b36443799 . _:b567468994 . . _:b567468995 . . . _:b567469004 . _:b567469005 . _:b36443792 . _:b567469006 . _:b36443778 "Differentiation of pancreatic CSCs was induced by culturing the cells under the differentiation-inducing condition (DMEM/F12 containing 10% FBS) for 2 weeks, as previously described [>>15<<]. Normal human IMR90 fetal lung fibroblasts [46, 47] were obtained from American Type Culture Collection and maintained in DMEM/F12 supplemented with 10% fetal bovine serum (FBS) and 100 units/mL penicillin and 100 \u03BCg/mL streptomycin. All" . . _:b567469007 . _:b567469000 . _:b567469001 . _:b36443793 . _:b567469002 . _:b567469003 . . _:b567469081 . _:b567469012 . _:b567469013 . _:b36443794 . . _:b567469014 . _:b36443809 "The role of KRas in the chemoresistance of pancreatic CSCs still remains to be explored, but a previous study showed that Ras inhibition using farnesylthiosalicylic acid sensitized pancreatic cancer cells to GEM [>>43<<]. It might therefore be tempting to speculate that KRas contributes to the chemoresistance of pancreatic CSCs as well through the activation of the JNK-ROS defense axis." . _:b567469015 . . _:b567469008 . _:b567469004 . _:b567469009 . _:b36443795 . . _:b567469010 . _:b567469011 . _:b567469020 . _:b567469025 . _:b567469021 . _:b36443804 . _:b567469022 . _:b567469023 . _:b567469016 . _:b567469017 . _:b36443805 . _:b567469018 . _:b567469019 . _:b36443791 . _:b36443767 "introduction" . _:b36443806 . _:b567468991 . _:b567469046 . _:b36443807 . _:b36443800 . _:b36443801 . _:b567469070 . _:b567469029 . _:b36443802 . . _:b36443803 . . _:b567469067 . _:b36443810 . _:b567469104 . . _:b36443808 . . _:b567468996 . _:b567469019 . _:b36443809 . _:b567469073 . . _:b36443810 . . _:b36443811 . . _:b567469024 . _:b567469064 . . . _:b567469006 . _:b36443795 . _:b36443772 . . _:b36443795 . _:b36443801 . _:b36443782 . . _:b36443790 . . _:b567469092 . . _:b567469093 . _:b567469094 . _:b567469095 . _:b567469012 . _:b567469088 . _:b567469013 . _:b567469089 . _:b567469102 . _:b567469090 . _:b567469091 . _:b567469100 . _:b567469101 . _:b36443772 . _:b567469102 . _:b567469103 . _:b567469096 . _:b567469097 . _:b567469045 . _:b567469098 . _:b567469099 . _:b36443771 . _:b567469108 . . _:b567469109 . _:b567469093 . _:b567469104 . _:b36443806 "Interestingly, these two studies showed that activated JNK was localized to mitochondria and that JNK inhibition led to accumulation of mitochondrial superoxide [40, >>41<<], pointing to the intriguing possibility that JNK may have a role in keeping the mitochondrial ROS level in check." . _:b567469105 . _:b567469106 . _:b36443788 . _:b567469107 . . . _:b36443783 . _:b567469060 . _:b567469061 . _:b567469062 . _:b567469061 . _:b567469063 . . _:b567469056 . _:b567469057 . _:b567468993 . _:b567469058 . _:b567469060 . _:b567468990 . _:b567469059 . _:b567469068 . _:b567469069 . _:b567469070 . _:b567469063 . _:b567469071 . _:b567469077 . _:b567469064 . _:b567469065 . _:b36443788 . _:b567469066 . . _:b567469067 . _:b567469062 . _:b567469076 . _:b567469023 . _:b567469077 . _:b567469078 . _:b567469057 . _:b567469079 . _:b567469072 . _:b567469103 . _:b567469073 . _:b567469074 . _:b567469056 . _:b567469075 . _:b567469084 . _:b567469085 . _:b36443788 . _:b567469086 . _:b567469059 . _:b36443799 "It has been demonstrated that CSCs have lower intracellular ROS levels and that the low ROS levels are associated with the radioresistance of CSCs [>>31<<, 32]. In contrast to radioresistance, however, the role of enhanced ROS defense of CSCs in chemoresistance has yet to be established. Significantly, lines of evidence suggest that ROS may participate in the cytotoxic mechanisms of" . _:b567469087 . _:b567469003 . _:b567469080 . _:b567469081 . _:b567469082 . _:b36443773 "CSCs in the poor prognosis of pancreatic cancer, suggesting that overcoming the chemoresistance inherently associated with pancreatic CSCs is apparently an indispensible step to improve overall management of pancreatic cancer [>>13<<, 14]. However, to date, the molecular basis on which pancreatic CSCs acquire resistance to conventional chemotherapeutic agents against pancreatic cancer has been poorly understood." . _:b567469058 . _:b567469083 . _:b567469069 . _:b36443804 . _:b567469068 . _:b567469071 . _:b567469070 . _:b567469065 . _:b567469010 . _:b567469064 . _:b567469067 . . _:b36443767 _:b36443768 . _:b36443767 _:b36443769 . _:b36443767 _:b36443770 . _:b567469066 . _:b36443767 _:b36443771 . _:b36443767 _:b36443772 . _:b36443767 _:b36443773 . _:b36443767 _:b36443774 . _:b567469077 . . _:b567469076 . . _:b567469079 . _:b567469098 . _:b36443780 "Normal human IMR90 fetal lung fibroblasts [46, >>47<<] were obtained from American Type Culture Collection and maintained in DMEM/F12 supplemented with 10% fetal bovine serum (FBS) and 100 units/mL penicillin and 100 \u03BCg/mL streptomycin." . . _:b567469078 . . _:b36443802 . _:b567469073 . _:b567469000 . . . . _:b567469072 . _:b567469053 . _:b567469075 . _:b567469008 . _:b567469074 . _:b567469085 . _:b567469084 . . . _:b567469087 . _:b567469080 . _:b567469086 . . _:b36443788 . _:b567469081 . _:b567469080 . _:b567469083 . _:b567468986 . . _:b567469082 . _:b567469002 . _:b567469093 . _:b36443805 "Interestingly, these two studies showed that activated JNK was localized to mitochondria and that JNK inhibition led to accumulation of mitochondrial superoxide [>>40<<, 41], pointing to the intriguing possibility that JNK may have a role in keeping the mitochondrial ROS level in check." . _:b36443789 "has a role in SP600125-mediated sensitization of PANC-1 CSLCs to 5-FU and GEM, we next examined the effect of the antioxidant N-acetylcysteine (NAC), which scavenges free radicals by increasing intracellular glutathione levels (GSH) [>>22<<]. NAC, which effectively suppressed the increase in intracellular ROS, inhibited sensitization of pancreatic CSCs to 5-FU and GEM by SP600125; NAC treatment significantly decreased the proportion of dead cells after treatment of PANC-1" . _:b567469092 . _:b567469083 . . _:b567468999 . _:b567469095 . _:b567469094 . _:b36443770 . _:b567469089 . _:b36443775 _:b36443776 . . _:b36443775 _:b36443777 . _:b36443775 _:b36443778 . _:b567469088 . _:b36443775 _:b36443779 . _:b36443775 _:b36443780 . _:b36443775 _:b36443781 . _:b567469091 . _:b567469043 . _:b567469090 . _:b567469101 . _:b567469027 . _:b567469100 . _:b36443772 . _:b567469103 . . . _:b567469102 . _:b567469097 . . . _:b567469096 . _:b567469094 . . . _:b567469099 . . _:b567469098 . _:b567469109 . _:b36443787 "We have recently demonstrated that the JNK signaling pathway is activated in self-renewing pancreatic CSCs compared with their differentiated counterparts [>>15<<]. Because our present data indicated that self-renewing pancreatic CSCs were more resistant to 5-FU and GEM (Figure 1; Supplementary Figure 1), we questioned whether JNK signaling is involved in the chemoresistance of pancreatic CSCs to" . _:b567469108 . . . _:b567469069 . . _:b567469105 . . . _:b567469104 . _:b567469061 "2"^^ . _:b36443784 . _:b567469107 . _:b567469060 "2"^^ . _:b567469039 . _:b567469106 . _:b36443768 . _:b567469038 . _:b567469063 "2"^^ . _:b567469062 "2"^^ . _:b567469095 . _:b567469057 "2"^^ . _:b567469048 . . _:b567469056 "2"^^ . _:b567469059 "2"^^ . _:b567469058 "2"^^ . _:b567469009 . . _:b567469022 . _:b567469069 "2"^^ . _:b567469068 "2"^^ . . _:b567469071 "2"^^ . . _:b567469065 . _:b567469086 . _:b567469070 "2"^^ . _:b567469065 "2"^^ . _:b567469064 "2"^^ . _:b36443772 . . _:b567469028 . _:b567469067 "2"^^ . _:b36443794 "Here in this study, we first found that pancreatic CSCs, which we have shown to have activated JNK [15], were highly resistant to 5-FU and GEM compared with their non-stem counterparts, essentially consistent with earlier reports [13, >>14<<]. We then demonstrated that inhibition of the activated JNK in pancreatic CSCs sensitized them to 5-FU and GEM and that JNK inhibition did so through elevation of the intracellular ROS level in combination with these chemotherapeutic" . . _:b567469066 "2"^^ . . . _:b567469077 "2"^^ . _:b36443802 . _:b567469105 . _:b567469035 . _:b567469076 "2"^^ . . . _:b567469079 "2"^^ . . _:b567469078 "2"^^ . _:b567469073 "2"^^ . _:b36443802 . _:b567469075 . _:b567469072 "2"^^ . . _:b36443785 "To examine whether this applies to the pancreatic CSCs we have established, we used PANC-1 CSLCs, CSCs derived from the pancreatic cancer cell line PANC-1 [>>15<<], and determined the sensitivity of self-renewing (undifferentiated) PANC-1 CSLCs and their serum-differentiated counterparts to 5-FU and GEM, which are current chemotherapeutic agents of choice for the treatment of pancreatic cancer." . _:b567468985 . . _:b36443777 "Unless otherwise indicated, these pancreatic CSCs were stably maintained and used for experiments under the monolayer stem cell culture condition, as previously described [>>15<<]. Briefly, cells were cultured on collagen-I-coated dishes (IWAKI, Tokyo, Japan) in the stem cell culture medium (DMEM/F12 medium supplemented with 1% B27 [Gibco-BRL, Carlsbad, CA, USA], 20 ng/mL EGF and FGF2 [Peprotech, Inc., Rocky Hill," . _:b567469075 "2"^^ . _:b567469041 . . _:b567469005 . _:b567469074 "2"^^ . . _:b36443775 "materials and methods" . _:b567469085 "2"^^ . _:b36443788 . _:b567469054 . _:b36443790 _:b36443804 . _:b36443788 "Since reactive oxygen species (ROS) have been implicated in the cytotoxicity of chemotherapeutic agents such as 5-FU and GEM [>>16<<\u201321], we examined the intracellular ROS levels of pancreatic CSCs after drug treatment." . _:b567469084 "2"^^ . _:b36443790 _:b36443805 . _:b36443790 _:b36443806 . _:b36443790 _:b36443807 . _:b36443790 _:b36443800 . _:b567469087 "2"^^ . . _:b36443790 _:b36443801 . _:b36443790 _:b36443802 . _:b36443790 _:b36443803 . _:b36443790 _:b36443796 . . _:b36443808 . _:b36443790 _:b36443797 . _:b567469086 "2"^^ . _:b36443790 _:b36443798 . _:b36443790 _:b36443799 . _:b36443790 _:b36443792 . _:b567469081 "2"^^ . _:b36443790 _:b36443793 . . _:b36443790 _:b36443794 . _:b36443790 _:b36443795 . _:b567469078 . _:b567469080 "2"^^ . _:b567468987 . _:b36443790 _:b36443791 . _:b567469083 "2"^^ . . _:b567469082 "2"^^ . . _:b567469093 "2"^^ . _:b567469092 "2"^^ . _:b567469063 . . _:b567469095 "2"^^ . _:b567469074 . _:b567469094 "2"^^ . _:b567469017 . _:b36443771 . . _:b567469089 "2"^^ . . _:b36443782 _:b36443788 . . _:b36443770 . _:b36443782 _:b36443789 . _:b567469088 "2"^^ . _:b36443782 _:b36443784 . _:b567469091 "2"^^ . _:b36443782 _:b36443785 . _:b36443782 _:b36443786 . _:b36443795 "Although JNK was originally identified as a positive regulator of cell death and overwhelming evidence does support the idea, JNK has also been shown to have a pro-survival function depending on the context [>>23<<\u201325]. Indeed, there are studies suggesting that the JNK pathway may play a positive role in chemoresistance of some cancer types [26]." . _:b36443782 _:b36443787 . _:b567469090 "2"^^ . _:b36443782 _:b36443783 . _:b36443811 . _:b36443790 _:b36443808 . _:b36443792 "Here in this study, we first found that pancreatic CSCs, which we have shown to have activated JNK [>>15<<], were highly resistant to 5-FU and GEM compared with their non-stem counterparts, essentially consistent with earlier reports [13, 14]." . _:b36443790 _:b36443809 . _:b567469101 "2"^^ . _:b36443790 _:b36443810 . _:b36443790 _:b36443811 . _:b567469015 . _:b567469100 "2"^^ . _:b567469103 "2"^^ . . _:b567469102 "2"^^ . _:b36443792 . _:b567469097 "2"^^ . _:b36443786 "We then conducted similar analyses using PSN-1 CSLCs, CSCs derived from another pancreatic cancer cell line PSN-1 [>>15<<]. The results indicated that the IC50 values of 5-FU and GEM for self-renewing PSN-1 CSLCs were again much higher than those for their differentiated counterparts [5-FU: approximately 1 \u03BCM and 0.3 \u03BCM for self-renewing and differentiated" . _:b567469096 "2"^^ . _:b567469071 . _:b567469099 "2"^^ . _:b36443798 "suggested that uPA and c-Met may also be involved in the chemoresistance of pancreatic CSCs, which is quite intriguing in light of the previous observations that JNK could function upstream and downstream of uPA and c-Met, respectively [>>28<<\u201330]. Although not pursued in the present study, investigation on the possible functional links among uPA, c-Met, and JNK may help better understand the whole picture of the molecular mechanism underlying the chemoresistance of pancreatic" . _:b36443769 . _:b567468997 . . _:b567469098 "2"^^ . .