Andrographolide Restores Sensitivity Of Gamma-secretase Inhibitors In T-cell Acute Lymphoblastic Leukemia

ObjectiveT-cell acute lymphoblastic leukemia (T-ALL) is a kind of hematological malignancy featuring malignant proliferation of immature T lymphoblast. The combined chemotherapy has been widely applied in clinic. However, the high recurrence rate and chemotherapy-resistant of T-ALL adding to its weak prognosis have been often observed. In recent years, therapeutical effect of y-secretase inhibitors (GSIs) to T-ALL has received much concern, because GSIs is a sort of small molecule compound with the effect to inhibit NOTCH1 activation, in addition the important role of abnormal activation of NOTCH1 in pathogenesis of T-ALL has been observed. However, there is no progress in clinical experiment for GSIs against T-ALL, as a result of the treatment resistence of T-ALL to GSIs and dosage-dependent gastrointestinal tract toxic reaction in clinic.This study focused on Andrographolide, a main active component of Andrographis paniculata(a traditional Chinese medicine), and used GSIs-resistent T-ALL in vitro to explore the effect and mechanism that Andrographolide inhibit GSIs-resistence and recover GSIs sensibility.And then by observing the effect of Andrographolide to peripheral blood lymphocyte from healthy subject,the security and selectivity of Andrographolide was determined.And in vivo,with the establishment of animal model of GSIs-resistence,the inhibition of Andrographolide to GSIs-resistence and recovery of Andrographolide to sensitivity of GSIs was detected,and the relaxation effect of Andrographolide combined with GSIs to GSIs-caused gastrointestinal tract toxicity was observed too.MethodsExplore the inhibition of Andrographolide to GSIs-resistence and effect of Andrographolide to peripheral blood lymphocyte of healthy subject. The T-cell acute lymphoblastic leukemia cell lines were selected (CRFF-CEM and Jurkat), the interference of Andrographolide to cell proliferation activity was observed by MTS, then the EC50 was detected. Use Andrographolide singly and Andrographolide combined with DAPT to interrupt CRFF-CEM and Jurkat.The cell proliferation activity was observed and cell proliferation curve was got, apoptosis was detected by Annexin-V/PI, and cell cycle was observed by PI staining. The peripheral blood lymphocyte of healthy subject was isolated,and with the dosage of 2,4,8 and 16μM Andrographolide, cell proliferation activity was explored by MTS, apoptosis was detected by Annexin-V/PI.Observe the effect mehamism of Andrographolide to GSIs-resistent T-ALL. Use Andrographolide singly, and Andrographolide combined with DAPT to interrupt CRFF-CEM and Jurkat.Then the expression of activated NICD in the NOTCH1 and pAkt, the significant protein in the PI3K/Akt/S6 pathway, were detected by western-blot. Use Andrographolide singly, and Andrographolide combined with DAPT to interrupt CRFF-CEM and Jurkat. Then the expressions of c-Myc and Mcl-1 the were detected by western-blot, the relative quantification expression of c-Myc and Mcl-1 were observed too by qRT-PCR. Use Andrographolide to to interfere CRFF-CEM and Jurkat,and by western-blot the expression of p105/p50, p65, IKKβ and IκBα in the NF-κB pathway were explored.Detect the effect mechnism that Andrographolide recover sensitivity of DAPT. Apply the inhibitor c-Myc only, and combination of c-Myc and DAPT to interfere CRFF-CEM and Jurkat. The cell proliferation activity was explored by MTS. Apoptosis was detected by Annexin-V/PI. Apply PI3K inhibitor PI-103 singly, and combination of PI-103 and DAPT to interrupt CRFF-CEM and Jurkat. By MTS the cell proliferation activity was explored, apoptosis was detected by Annexin-V/PI. The expession of NICD, c-myc, p-Akt and pS6 was explored by western-blot and cell proliferation activity and apoptosis were observed after combination use of 10058-F4, PI-103 and DAPT to interrupt CRFF-CEM and Jurkat. The Jurkat was violated by lentivirus packaging shRNA clone of MCL1, and the transcriptional activation and expression of Mcl-1 were silenced, then the influence by DAPT to cell proliferation activity and apoptosis of Jurkat was explored.Observe Andrographolide inhibited GSIs-resistence and relaxed GSIs-caused gastrointestinal tract toxic reaction. Give BAlB/c nude mice cell suspension (subcutaneous injection) to make GSIs-resistent T-ALL model. Then the Andrographolide 200mg/kg and DAPT 25mg/kg separately were given (intragastric administration), and gave the combination of Andrographolide and DAPT orally, and record the change of tumor volume and calculate tumor inhibition rate after 20days. The expression of Ki-67 and Caspase-3 was detected by immunohistochemistry. Explore NICD, pAkt, p65, c-Myc, pS6 and Mcl-1 protein expression by western-blot. Use the C57BL/6 mice,25mg/kg, DAPT, 100mg/kg DAPT and 200mg/kg Andrographolide was given (intragastric administration) separately, and the 200mg/kg Andrographolide combined with 25mg/kg DAPT were given by gavage, after 7 days, the duodenum of C57BL/6 mice was got, and pathologic change of epithelium goblet cell of intestinal tract was observed.ResultsAndrographolide can inhibit evidently the cell proliferation of CRFF-CEM (EC50=2.604μM) and Jurkat (EC50= 3.782μM). While Andrographolide derivative with Lactone removed had no obvious effect on cell activity. There was no remarkable effect observed by Andrographolide on cell activity, apoptosis and cell cycle of CRFF-CEM and Jurkat. Andrographolide can inhibit cell proliferation activity of DAPT-resistent CRFF-CEM and Jurkat, induce apoptosis and block cell cycle, and all of these effects can be more obvious under combined use of Andrographolide and DAPT. There was no peripheral blood lymphocyte apoptosis of healthy subject observed after giving Andrographolide, furthermore it can facilitate cell proliferation.A suppression effect to NICD was showed after giving Andrographolide, and a down-regulation effect on expression of activated key protein pAkt and pS6 in the PI3K/Akt pathway was observed too. A down-regulaton effect on overexpression of c-Myc and Mcl-1 induced by FBXW7-caused mutaion Andrographolide, and a suppression to transcriptional activation of MYCand MCL1 were detected. And Andrographolide can activate the suppression effect of DAPT to c-Myc. Andrographolide suppressed NF-κB activation, and regulated down p65 expression relative to p105/p50.In addition, Andrographolide inhibited activated expression of IKKP, but had no influence in expression of IκBα.The suppression to cell proliferation of CRFF-CEM and Jurkat after giving c-Myc inhibitor 10058-F4 and PI3K inhibitor PI-103 was showed. While combination use of10058-F4, PI3K and DAPT can’t recover DAPT-induced suppression to cell proliferation activity and apoptosis. The combined use of 10058-F4,PI-103 and DAPT showed a strong inhibition effect to PI3K/Akt/S6 pathway, c-Myc and NICD.And relative to single application of 10058-F4 or PI-103,the combination of them exhibited a stronger ability to suppress cell proliferation and induce apoptosis. While,the combined use of 10058-F4, PI-103 and DAPT demonstrated a limited effect on the recovery of DAPT’suppressing cell proliferation and inducing apoptosis. DAPT exhibited a certain effect on suppression and induing apoptosis to Jurkat with Mcl-1 expression silenced.Andrographolide suppressed BAlB/c nude mice the growth of subcutaneously transplanted tumor, while DAPT showed no ability to inhibit tumor growth, however, combined use of them demostrated a stronger ability to suppress tumor growth. Andrographolide enhanced the expression of apoptin Caspase-3 by suppressing expression of ki-67, a protein who can illustrate cell proliferation activity. Furthermore, combination use of them showed a stronger effect to suppress expression of Ki-67 and enhance expression of Caspase-3. DAPT demostrated a strong suppression effect to NICD in vivo.And Andrographolide combined with DAPT keep a same effect on suppression to expression of NICD. pAkt, p65, c-Myc,pS6 and Mcl-1 as it did in vitro. The remarkable increase in goblet cell of duodenum of C57BL/67 days aftergiving 1 OOmg/kg DAPT was observed, while there was no significant change at the dosage of 25mg/kg DAPT, and there was no obvious pathologic change too with combined use of 200mg/kg Andrographolide and 25mg/kgDAPT.Conclusion:Andrographolide suppressed GSIs-resistent cell proliferation, induced apoptosis, blocked cell division cycle, and showed a certain effect to recover sensitivity of DAPT. And the -OH on lactone ring of Andrographolide played a very significant role in the activity of Andrographolide.And There was no peripheral blood lymphocyte apoptosis of healthy subject observed after giving Andrographolide, on the contrary, it can facilitate cell proliferation.Andrographolide exhibited a certain effect on suppression to activation of NOTCH1 of cell, furthermore, it also demonstrated a evident suppression to activation of NOTCH1 of GSIs-resistent cell, which might be caused by following mechanisms:(1) Andrographolide regulated down overactivation of PI3K/Akt induced by the lack or inactivation of PTEN. (2) Andrographolide suppressed overexpression of c-Myc induced by mutation of FBXW7, and it activated DAPT’down-regulation to c-Myc. (3) Andrographolide blocked the apoptosis escape induced by FBXW7’mutation-caused uncontrollability of Mcl-1.(4) Andrographolide suppressed activation of NF-κB pathway.To singly suppress c-Myc or PI3K/Akt pathway can not recover sensitivity of DAPT, and there was no obvious effect to recover sensitivity of DAPT when suppressing both of c-Myc and PI3K/Akt pathway.Andrographolide showed activities of anti-T-ALL and recovery to activity of DAPT in vivo. Andrographolide suppressed tumor cell proliferation and induced apoptosis in vivo, and showed a same effect to the proteins which plays a significant role in occurrence of T-ALL as it did in vitro. Andrographolide combined with low dosage of DAPT demonstrated a relief to dose-dependent gastrointestinal tract toxic reaction by DAPT.