We jointly analyzed the changes in cell cycle arrest and distribution, the accumulation of subphase cells, apoptosis, and proliferation in A549 cells treated with Saikosaponin D (Ssd) and JNK inhibitor SP600125 alone or in combination. Our results indicated that cell cycle arrest at G0/G1, S, and G2/M phases was coupled with the accumulation of subG1, subS, and subG2 cells, corresponding to early apoptosis, DNA endoreplication, and later inhibitory proliferation, respectively. Analyzing the expression of 18 cell cycle regulatory genes and JNK and phosphorylated JNK (pJNK) levels revealed an enhancement in these factors by Ssd. Additional SP600125 weakened or eliminated the Ssd-induced increase of these factors except that p53/p21 and Rassfia levels were further improved. Ingenuity Pathway Analysis (IPA) of the interactions of these factors revealed a negative synergistic effect on apoptosis while a positive synergistic effect on proliferative inhibition of the two drugs: (1) Ssd induced apoptosis via the activation of two axes, TGFα-JNK-p53 and TGFα-Rassfia-Mst1. By eliminating the Ssd-induced increase of JNK/pJNK, additional SP600125 weakened the Ssd-induced apoptotic axis of TGFα-JNK-p53 and simultaneously abolished Ssd-induced apoptosis; (2) Ssd inhibited proliferation by the activation of two axes, TGFβ-p53/p21/p27/p15/p16 and TGFα-Rassfia-cyclin D1. By improving the Ssd-induced increase of p53/p21 and Rassfia, additional SP600125 enhanced the two axes of Ssd-induced inhibitory proliferation. Analyzing JNK/pJNK, p53, phospho-p53, and TNF-α levels revealed an opposite association of JNK/pJNK with p53 while consistent with phospho-p53 and TNF-α, which supported the proposals that JNK/pJNK negatively regulated p53 level, while it mediated p53 phosphorylation to transcriptionally activate TNF-α expression of apoptotic gene and trigger apoptosis. With the multiple roles, JNK/pJNK forms a synergetic and antagonistic feedback loop with phospho-p53/p53. Within the feedback loop, (1) Ssd-induced apoptosis depended on JNK/pJNK activities mediating phospho-p53 that activated TNF-α expression; (2) by weakening the negative regulation of JNK/pJNK in p53, SP600125 enhanced p53 level and the Ssd-induced inhibitory proliferation axes of TGFβ-p53/p21/p27/p15/p16. The results indicated the central coordinating roles of the feedback loop in the synergistic and antagonistic effects of the two drugs in A549 cells and provided a rationale for the combination of Ssd with SP600125 in the treatment of lung cancer.
Keywords: Ingenuity Pathway Analysis; SP600125 of JNK inhibitor; Saikosaponin D; cell cycle regulatory gene; feedback loop involving phospho-p53/p53 and JNK/pJNK; lung cancer A549 cells.