Research On The Role Of Protein Kinase C In Tumor Repopulation And The Underlying Mechanism

Tumor cell repopulation has been recognized as a major cause of tumor relapse in treating cancers after radiotherapy or chemotherapy. In order to address the problem of tumor repopulation, our previous studies have demonstrated that dying tumor cells could generate potent growth-stimulating signals to stimulate the proliferation of neighboring living tumor cells after radiotherapy or chemotherapy. And activated caspase 3, which is a key executioner in apoptosis, is involved in the growth stimulation. Activated caspase 3 could regulate the activation of Ca2+-independent phospholipase A2(i PLA2) to stimulate the release of arachidonic acid, resulting in increased prostaglandin E2(PGE2) production; and plenty of previous studies have demonstrated that PGE2, a potent mitogen, could finally stimulate the proliferation of neighboring live tumor cells. However, in addition to i PLA2, we deduce that caspase 3 can regulate the process of tumor repopulation via activating several other targets, such as protein kinase C(PKC). PKC is kinase family including at least 11 subtypes. Different subtypes were considered to have different functions; and some of these subtypes, such as PKCδ, could be cleaved and activated by caspase 3 and have a major role in stimulating cell growth. Therefore, the present study is intended to determine the role of protein kinase C in pancreatic cancer and colonic cancer tumor repopulation and the underlying molecular mechanism.After radiotherapy, a large amount of tumor cells underwent apoptosis, but a smaller number of surviving tumor cells would proliferate at a markedly accelerated pace. This is thought to be a major cause of tumor relapse. We constructed and optimized an in vitro model of pancreatic cancer and colonic cancer cell growth after radiotherapy. In this model, a large number of tumor cells were treated with X-ray and used as “feeder cells”, and a much smaller number of living, luciferase-labeled tumor cells were seeded onto the large amount of unlabeled feeder cells and used as “reporter cells”; and growth of the living cells was measured over time as a gauge of tumor repopulation. Our results show that a significant number of apoptotic cells increased in pancreatic cancer cell Panc1 and colonic cancer cell HT29 after radiation; and these apoptotic Panc1 or HT29 cells significantly stimulated the proliferation of living tumor cells. Importantly, we identified caspase s 3 and 7 and PKCδ as integral mediators in the process of dying pancreatic cancer and colonic cancer cell stimulation of living tumor cell growth. Our results show that the cleaved caspases 3 and 7 and PKCδ protein levels were significantly increased in irradiated, apoptotic Panc1 and HT29 cells. In order to demonstrate the importance of caspases 3 and 7 and PKCδ, we found a significant decrease in the growth of living tumor cells when apoptotic Panc1 or HT29 cells stably transduced by dominant-negative mutants of caspase 3, caspase 7, or PKCδ were used as feeders instead of apoptotic wild-type tumor cells. Moreover, the role of PKCδ in the growth stimulation of living tumor cells was further confirmed using a pan PKC inhibitor GF109203 x and a specific PKCδ inhibitor, Rottlerin. Moreover, PKCδ cleavage was significantly attenuated in both DN_C3 and DN_C7 transduced Panc1 cells following radiation, suggesting that PKCδ was the downstream effector of caspases 3 and 7 in this process of dying tumor cell stimulation of living tumor cell growth.To further investigate the underlying signaling mechanism of the PKCδ-mediated growth stimulatory effect on living tumor cells, we examined the protein levels or phosphorylation status of several signaling molecules via Western blot. We found significantly increased phosphorylation of Akt, p38 mitogen-activated protein kinase(MAPK) and c-Jun N-terminal kinase/stress-activated protein kinase(JNK1/2) in the irradiated Panc1 and HT29 cells. Both Akt and p38 MAPK phosphorylation were significantly attenuated in DN_PKCδ transduced Panc1 cells following radiation, but only Akt in DN_PKCδ transduced HT29 cells. This result suggests that there may be subtle difference in the downstream signaling molecules between these two cancer cells, and Akt may be more extensively involved in this process of PKCδ-mediated dying tumor cell stimulation of living tumor cell growth.In HT29 cells, we pretreated apoptotic HT29 cells with PI3K/Akt inhibitor LY294002 and found the growth stimulation of living HT29 cells was significantly attenuated when Akt activation had been inhibited. This result further confirms the importance of Ak t in this process of dying tumor cell stimulation of living tumor cell growth and indicates that cellular signaling from caspase 3/7-PKCδ-Akt is crucial to the repopulation in colonic cancer cell HT29 after radiotherapy.In an effort to explore downstream growth factors of Akt, we examined the protein levels of vascular endothelial growth factor(VEGF) in Panc1 cells via Western blot. We found that the protein levels of VEGF were significantly increased in Panc1 cells after radiation but attenuated by the PI3K/Akt inhibitor LY294002. Meanwhile, the protein level s of VEGF receptor i.e. Flk-1 were extremely high in Panc1 cells. We suggest that cellular signaling from caspase 3/7-PKCδ-Akt-VEGF is crucial to the repopulation in pancreatic cancer cell Panc1 after radiotherapy.This report suggests that cellular signaling from caspase 3/7-PKCδ-Akt-VEGF is crucial to the tumor repopulation after radiotherapy; and provides new clues and targets to improve the efficacy of cancer radiotherapy and to develop new pharmaceuticals.