| Objective:The poor efficacy of systemic chemotherapy has become the bottleneck of further improving the prognosis of hepatocellular carcinoma(HCC) patients. As a pleiotropic cytokine with biological activities of antiproliferative, proapoptotic and immunomodulatory effect, interferon-α (IFN-α) has been reported to be effective in postponing recurrence and improving overall survival in patients after curative resection of HCC. However, despite the favorable effect in a large proportion of patients, a significant group of HCC patients show initial or acquired resistance to this therapy. Tumor resistance to IFN-α is hence a major hurdle in the management of HCC and strategies help to improve the efficacy of IFN-α is greatly needed to achieve a better control of the disease. Our study demonstrated that low concentrations of Aspirin (1mM) did enhance IFN-α-induced apoptosis and antiproliferation effect of HCC, which may lay the groundwork for possible future clinical evaluation of this combination.Methods:(1) Four groups of BEL-7402cells were divided:the control group, the5000U/ml IFN-α treatment group, the1mM Aspirin treatment group,5000U/ml IFN-α+1mM Aspirin treatment group;(2) MTT and flow cytometry were used respectively to measure the cell proliferation and apoptosis after the cell were treated with the alone or combined drugs. The expressions of the apoptosis-related proteins were detected by western blot.(3) To further investigate whether low concentration of Aspirin enhanced the effect of IFN-α through STAT1or STAT3signal transduction, we measured the change of JAK-STAT1/3signaling pathway in response to IFN-α stimulation with or without Aspirin at various time points. Besides, real time PCR was further used to examine whether Aspirin increased expression of p-STAT1could influence the regulation of ISGs after BEL-7402cells were treated with IFN-α, low concentration of Aspirin or the combination.Results:1. Aspirin promoted the IFN-α induced inhibition of HCC cell growth Cell viability was measured at24,48and72h after treatment. At the concentrations tested, IFN-α (5000IU/ml) caused major growth inhibition as a single agents, while low concentrations of Aspirin (1mM) caused growth inhibition after48h treatment (P<0.01). Combined use of IFN-α and Aspirin significantly inhibited cell proliferation than either agent alone (P<0.01).2. Aspirin enhanced IFN-α induced apoptosis of HCC cells At the concentrations tested, both IFN-α (5000IU/ml) and Aspirin (1mM) could elicit a small number of cell apoptosis as single agent, but when they were used together, apoptosis of BEL-7402cells were significantly increased (P<0.01), implying a synergetic effect of IFN-α and Aspirin on apoptosis of HCC cells.3. Aspirin enhanced IFN-α induced apoptosis via caspase cascade proteinsWe further analyzed the expression of biological markers of apoptosis. Though Aspirin (1mM) did not induce the expression of cleaved caspase-3, however, when Aspirin and IFN-α were used together, they did result in marked cleavage of caspase-3, which was much more obvious than IFN-α (5000IU/ml) treatment alone (P<0.01). Furthermore, combined use of IFN-α and Aspirin also resulted in enhanced caspase-9cleavage than IFN-α or Aspirin treated group (P<0.01,), while the change of caspase-8activity was not detected in the same treated samples (P>0.05). Besides, the expression of Bcl-2, Bcl-xl and Mel-1were not changed in IFN-α or Aspirin treated group, as well as in combined use group (P>0.05). However, the expression of Bax in IFN-a or Aspirin treated group was significantly enhanced. And when IFN-a were combined with Aspirin, the expression of Bax was further elevated than either treatment alone (P<0.01)4. Aspirin promoted the expression of phosphorylated and total STAT1induced by IFN-aWhen IFN-α was combined with Aspirin, the duration and level of p-STAT1were significantly enhanced than IFN-α treatment alone (P<0.01), while theexpression of p-STAT3was not changed significantly (P>0.05). The synergetic effect of Aspirin on IFN-α induced phosphorylation of STAT1still existed18h later (P<0.05), and total STAT1also began to increase after4h stimulation and was significantly enhanced18h later (P<0.01), but the expression of IFN-α induced p-STAT3was still not changed significantly after18h stimulation of Aspirin (P>0.05). Total STAT3expression was still not influenced after18h combined treatment of IFN-α and Aspirin (P>0.05)5. Aspirin enhanced expression of IFN-α-induced p-STAT1was mediated through the activation of JAK2IFN-α alone significantly enhanced the phosphorylation of JAK2, and when it was combined with Aspirin, the phosphorylation of JAK2was further elevated(P<0.01). The phosphorylation of TYK2was significantly inhibited when IFN-α was combined with Aspirin, compared to IFN-α treatment alone (P<0.01).However, the phosphory-lation of JAK1was not detected when BEL-7402cells were stimulated by IFN-α with or without Aspirin (P>0.05). Besides, after blocking the activity of JAK2by100uM AG490, the phosphorylation of STAT1and STAT3induced by combined use of IFN-α and Aspirin was significantly decreased, compared to groups in which JAK2activity was not inhibited (P<0.01).6. Aspirin influenced the IFN-α induced transcriptions of ISGs mRNAWe next examined whether Aspirin increased expression of p-STAT1could influence the regulation of ISGs. Real Time PCR analysis revealed that low concentrations of Aspirin increased the IFN-α induced transcriptions of XAF-1(P<0.05), and inhibited the transcriptions of GIP3. Conclusion:1. Low concentration of Aspirin enhances IFN-α-induced growth inhibition and apoptosis of HCC via JAK2/STAT1pathway. These results lay the groundwork for possible future clinical evaluation of this combination, and may provide a new potential therapeutic strategy for patients with HCC.2. The activation of JAK2/STAT1pathway induces the upregulation of proapoptotic ISGs of XAF1and downregulation of antiapoptotic ISGs of G1P3, which in turn promote the expression of Bax and activation of caspase-9and caspase-3, thereby sensitize more HCC cells to IFN-α induced apoptosis. |
PDF Full Text Request