PCT3, a Combination of Active Steroidal Saponins from the Paris Forrestii (Takht.) H. Li, Displays Potent Activity Against Acute Myeloid Leukemia By Suppressing the PBX1/RNF6/AKT/mTOR Signaling Pathway

Acute myelogenous leukemia (AML) is a class of malignancy derived from the myeloid line of aberrant progenitor/stem cells from bone marrow. AML is incurable and the five year rate of the event free survival (EFS) remains less than 20% even with the intensive chemotherapy in combination with autologous hematopoietic stem cell transplantation. Therefore, it is urgent to find novel drugs to improve the outcome of AML patients. Paris forrestii (Takht.) H. Li is a traditional herb medicine specifically produced in the Yu'nan and Tibet regions of China and it is widely used for the inflammation and coagulation among local people. In the study to identify natural products for cancer treatment, we isolated and evaluated the active ingredients from Paris forrestii (Takht.) H. Li and found that PCT3, a combination of steroidal saponins, displayed potent anti-AML activity. The high performance liquid chromatography/mass spectrometry (HPLC/MS) assay isolated and identified 10 steroidal saponins (SPs). In the preliminary study, each individual SP displayed limited cytotoxicity to AML cell lines K562 and HL-60. However, when a random combinations of the SPs were tested, it turned out that PCT3 (mainly composed of Polyphyllin I and III) was the most potent one in inhibiting AML cell prolipferation. The IC50 of PCT3 on K562 was 2.935 ± 0.772 μg/ml within 24 hrs. Moreover, PCT3 induces potent AML cell apoptosis in a concentration-dependent manner. At the ranges of 0 to 8μg/ml, PCT3 induces apoptosis of selected AML cell lines, including K562, HL-60, HT-93 and KG-1 as evidenced of the cleavage and activation of Caspase-3 and PARP. Consistent with this finding, PCT3 downregulated oncoproteins such as Bcl-2 and Mcl-1 but upregulated pro-apoptotic signaling proteins including Bax. The anti-AML activity of PCT3 was further evaluated in AML xenografts in nude mice derived from K562 cells. Oral administration of PCT3 at 100 mg/kg for a continuous 14 days markedly delayed AML tumor growth but had no effects on mice body weight. These results thus suggested that PCT3 displayed potent anti-AML activity in both molecular, cellular and nude mice levels. To understand the underlying molecular mechanisms PCT3 kills AML cells, we first evaluated the AKT/mTOR signaling pathway, a central node of AML cell proliferation and AML cell survival. To our expectation, PCT3 suppressed the activation of the AKT/mTOR signals. PCT3 inhibited the phosphorylation of AKT and mTOR, but had no effects on their total protein expression levels. Consistently, 4E-BP1 and p70S6K, two typical downstream proteins of the AKT/mTOR pathway, were also inhibited by PCT3 in a concentration-dependent manner. Moreover, we found that PCT3 inhibited the expression of the PBX1, a key transcription factor in AML (Xu X et al, J Biol Chem, 2016). PBX1 promotes leukemia cell proliferation by promoting the transcription of RNF6, a ring finger protein. Interestingly, the PBX1/RNF6 axle upregulated the activation of the AKT/mTOR signaling pathway. Therefore, we found that PCT3 displayed potent anti-AML activity by suppression the PBX1/RNF6/AKT/mTOR signaling pathway. Given its tolerable toxicity and anti-AML activity, PCT3 could be developed for AML treatment.