Apoptosis of T-acute lymphoblastic leukemia cells is induced by the combination of PI3K and autophagy inhibitors

doi:10.21203/rs.3.rs-1866537/v1

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Apoptosis of T-acute lymphoblastic leukemia cells is induced by the combination of PI3K and autophagy inhibitors

https://doi.org/10.21203/rs.3.rs-1866537/v1

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Background: The PI3K/Akt signaling pathway plays an important role in the occurrence and development of tumors, and various PI3K inhibitors have been reported in antitumor. However, PI3K inhibitors can induce autophagy and activation of autophagy can promote drug resistance of tumor cells or improve cell survival. Therefore, the present study aimed to investigate the effects of the PI3K inhibitor PX-866 or PI-103 combined with the autophagy inhibitor 3-methyladenine (3-MA) on the apoptosis of acute T-acute lymphoblastic leukemia cells lines CCRF-CEM and Jurkat cells.

Methods and results: We found PX-866 and PI-103 treatment significantly reduced cell viability whilst also greatly increasing apoptosis in CCRF-CEM and Jurkat cells. The efficacy of this treatment was further enhanced when combined with 3-MA. The phosphorylation levels of AKT and mTOR in CCRF-CEM and Jurkat cells were significantly suppressed by the introduction of PX-866 or PI-103, the effects of which were reversed by co-treatment with 3-MA. In addition, the expression of LC3, ATG5, ATG12 and Beclin-1 were found to be significantly upregulated by PX-866 or PI-103 treatment and were markedly downregulated by the co-administration of 3-MA. TEM results revealed that the number of autophagic vacuoles in CCRF-CEM and Jurkat cells was increased by PX-866 and PI-103 treatment, which was reversed by 3-MA co-treatment.

Conclusions: The combination of PI3K and autophagy inhibitors reduced cell viability and induced apoptosis in T-acute lymphoblastic leukemia cells. This discovery provided a new way to improve the effectiveness of anti-tumor strategies in T-cell acute lymphoblastic leukemia.

PI3K inhibitor

autophagy inhibitor

T-acute lymphoblastic leukemia

apoptosis

T-cell acute lymphoblastic leukemia(T-ALL) is one of hematological malignancies [1] and characterized by high rates of relapse and poor prognosis [2]. Mutations of PIK3CA key components in the PI3K/AKT/mTOR signaling pathway have been previously reported in T-ALL cells [3, 4]. Therefore, PI3K/AKT/mTOR signaling becomes a critical pathway for the investigation of the pathogenesis of T-ALL [5].

PI3K/AKT/mTOR signaling has been reported that serves an important role in cellular biological processes, such as cell proliferation, apoptosis, cell cycle progression and angiogenesis [6]. A number of molecules (e.g., PI-103 and PX-866) targeting the PI3K/AKT/mTOR pathway have been approved to progress to clinical trials and provide novel therapies of solid tumors including liver cancer and gastric cancer [7, 8].

As a semi-synthetic derivative of wortmannin [9], PX-866 binds to PI3K by covalent bonding and becomes the only irreversible PI3K inhibitor that is undergoing clinical trials recently[10, 11]. PX-866 treatment has been previously reported to suppress proliferation of cancer cells[12, 13]. In addition, PX-866 can inhibit the invasive capabilities of glioblastoma cells and suppress angiogenesis [14]. Previous PDX models revealed that after PX-866 treatment, the growth of glioblastoma xenograft tumors was significantly inhibited, where the median survival times of animals with xenografts were dramatically prolonged [15]. In the other hand,PI-103 is a novel dual-target inhibitor that targets both PI3K and mTOR, which can supress the proliferation of malignant tumor cells(e.g., hepatocellular carcinoma, and breast cancer) [16, 17]. In addition, PI-103 has demonstrated promising antitumor efficacy [18, 19] and low levels of cytotoxicity against a number of metastatic cancer types, including lymphoma, melanoma and lung cancer[20] .

PI3K inhibitors can induce cell cycle arrest, facilitate apoptosis and suppress cell proliferation [21], whilst mTOR inactivation can induce autophagy[22]. Therefore, inhibitors of the PI3K/mTOR pathway not only induces apoptosis but can also activate autophagy, which further affects the activity of SGK3 which is a hepatocellular carcinoma stem cell promoter. As a consequence, the survival of hepatocellular carcinoma cells is prolonged and the antitumor efficacy of PI3K/mTOR inhibitors is weakened [23].

The present study aims to explore the effects of the combination of a PI3K inhibitor and an autophagy inhibitor on the apoptosis and viability of acute lymphoblastic leukemia T lymphocytes. It is hoped that results from the present work can provide a theoretical foundation for the clinical treatment of T-ALL.

Cells and treatments.Human acute T-acute lymphoblastic leukemia cells, namely CCRF-CEM and Jurkat cells, were purchased from BeNa Culture Collection; Beijing Beina Chunglian Biotechnology Research Institute. They were cultured in RPMI-1640 medium (Nanjing KeyGen Biotech Co., Ltd.) supplemented with a mixture of penicillin and streptomycin (Beijing Solarbio Science & Technology Co., Ltd.) and 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37˚C in a humidified atmosphere containing 5% CO₂.

Drugs and groupinp.PX-866 (cat. no. HY-N6775), PI-103 (cat. no. HY-10115), and 3-methyladenine (3-MA, cat. no. HY-19312) were obtained from MedChemExpress. In total, the following five groups were established for the vitro experiments: i) Control (PBS); ii) PX-866 (5 µM dissolved in PBS); iii) PI-103 (5 µM dissolved in PBS); iv) PX-866 + 3-MA (5 µM PX-866 and 10 mM 3-MA dissolved in PBS); and v) PI-103 + 3-MA (5 µM PI-103 and 10 mM 3-MA dissolved in PBS) all at 37˚C for 24 h.

Cell Counting Kit-8 (CCK-8) assay.Briefly, the cells (1.0x10⁴ cells/well ) were added to a 96-well plate (cat. no. 3599; Corning, Inc.) and mixed with 10 µl CCK-8 solution (cat. no. KGA317; Nanjing KeyGen Biotech Co., Ltd.) and incubated at 37˚C for 2 h. Absorbance in each well was then measured at 450 nm by using a microplate reader (J&H Technology Co., Ltd.) ( http://www.liankebio.com/ )to determine cell viability, all operations follow the reagent and instrument instructions.

Flow cytometry for the analysis of apoptosis.Cells were seeded into six-well plates at densities of 2x10⁵ cells/well for 24 h at 37˚C and then resuspended in 500 µl annexin V binding buffer containing 5 µl of PI and 5 µl of annexin V-FITC [cat. no. AP101-100-kit] at 37˚C in the dark for 45 min. Subsequently, the 1x10⁵ cells were subjected to flow cytometry version 1.2.5 vendor Agilent to determine the apoptotic rate of each group(The third and fourth quadrants represent the influence of cell apoptosis.).

Western blot analysis. Total proteins were obtained from the cells by using RIPA lysis buffer (Applygen Technologies.) and quantified using a BCA kit (CoWin Biosciences). The protein samples were then transferred onto PVDF membranes. The membranes were then incubated with primary antibodies against AKT (cat. no. 44-609G; Invitrogen.), p-AKT (cat. no. 44-621G; Invitrogen.), mTOR ( cat. no. PA5-34663; Invitrogen.), p-mTOR (cat. no. 44-1125G; Invitrogen.), ATG5 ( cat. no. PA5-35201; Invitrogen.), ATG12 (cat. no. MA5-27801; Invitrogen.), Beclin-1 (cat. no. MA5-15825; Invitrogen.), GADPH (cat. no. 39-8600; Invitrogen.), LC3 I/II ( cat. no. PA5-22731; Invitrogen.) overnight at 4˚C. The membranes were incubated with the secondary antibody ( cat. no. 62-8420; Invitrogen.) for 60 min at room temperature. The bands were quantified using the ImageJ software V1.8.0.112 .

Transmission electron microscopy (TEM).Cells were collected by centrifugation at 750 x g for 5–10 min, at room temperature. The supernatant was then discarded and the cells were fixed with 2.5% glutaraldehyde, at 4˚C for 24 h. Next, the cells were washed with pre-cooled PBS buffer and dehydrated with an ascending gradient of alcohol and acetone. The samples were incubated with paraffin at room temperature overnight for embedding. Then the samples were sectioned at 70 nm by using an ultramicrotome and subsequently subjected to dual staining with 2% uranyl acetate and lead citrate at room temperature for 15 min. Finally, the slides were observed under a transmission electron microscope (magnification x20,000) (FEI Tecnai Spirit 12.).

Statistical analysis.Data were analyzed by using the Graphpad software (Graphpad Software 7.0, Inc.) and presented as the mean ± SD. Data were compared among groups using one-way ANOVA followed by Bonferroni corrections. All experiments were repeated three times independently. P < 0.05 was considered to indicate a statistically significant difference.

Effects of PI3K inhibitor combined with 3-MA on the cell viability of CCRF-CEM and Jurkat cells. The viability of CCRF-CEM and Jurkat cells after treatment with PX-866 and PI-103 cells was first measured. As shown in Fig. 1A and B, compared with control group, PX-866 or PI-103 treatment significantly reduced the viability of CCRF-CEM and Jurkat cells. In addition, 3-MA was added to CCRF-CEM and Jurkat cells alongside PX-866 or PI-103. The results showed that compared with that in the PI303 or PX-866 treatment group, the co-administration of 3-MA significantly suppressed the cell viability of CCRF-CEM and Jurkat cells (Fig. 1A and B).

Effects of the PI3K inhibitor combined with 3-MA on the apoptosis of CCRF-CEM and Jurkat cells.In accordance with the observations from cell viability experiments ( Fig. 1), the apoptosis rates of CCRF-CEM and Jurkat cells after treatment with PX-866 and PI-101 were measured then. As shown in Fig. 2A and B, compared with control, PX-866 or PI-103 treatment obviously increased the apoptosis of CCRF-CEM and Jurkat cells. Furthermore, the apoptosis of CCRF-CEM and Jurkat cells co-treated with 3-MA was markedly potentiated (Fig. 2A and B).

PI3K/AKT/mTOR pathway was affected by the PI3K inhibitor combined with 3-MA.

Since both PX-866 and PI-103 induced apoptosis in T-ALL cells, the expression of proteins related to the PI3K/AKT/mTOR pathway was then measured. As shown in Fig. 3A and B, treatment with PX-866 or PI-103 evidently reduced the phosphorylation levels of AKT and mTOR in both CCRF-CEM and Jurkat cells compared with the control group. The levels of AKT and mTOR phosphorylation were then measured in CCRF-CEM and Jurkat cells following additive treatment with 3-MA and PX-866 or PI-103. We discovered co-administration with 3-MA significantly reversed the phosphorylation of AKT and mTOR in CCRF-CEM and Jurkat cells (Fig. 3A and B). These results suggested that PX-866 or PI-103 could inhibit the activation of the PI3K/AKT/mTOR pathway in CCRF-CEM and Jurkat cells. By contrast, 3-MA inhibited the activation of this pathway.

Autophagy is altered by the PI3K inhibitor combined with 3-MA. The role of autophagy in apoptosis induced by PX-866 and PI-103 was next explored. The LC3II/I ratio was found to be significantly elevated in CCRF-CEM and Jurkat cells after the introduction of PX-866 or PI-103 (Fig. 4A and B). However, after 3-MA co-treatment, this increase in the LC3II/I ratio was markedly reversed (Fig. 4A and B).Meanwhile, treatment with PX-866 or PI-103 significantly increased the expression levels of ATG5, ATG12 and Beclin-1. And co-administration of 3-MA distinctly reversed this increase in the expression of ATG5, ATG12 and Beclin-1 (Fig. 4A and B).

Effects of the PI3K inhibitor combined with 3-MA on the number of autophagosomes formed. As shown in Fig. 5A and B, PX866 or PI-103 treatment obviously increased the number of autophagosomes in CCRF-CEM and Jurkat cells compared with the control group, suggesting that the autophagy was induced by PX-866 and PI-103. On the other hand, co-administration with 3-MA markedly decreased the number of autophagosomes compared with PI3K inhibitor treatment alone (magnification, x20,000) (Fig. 5A and B).

Taken together, these results indicate that autophagy serves a key role in the apoptosis of acute T-cell lymphoblastic leukemia cells induced by PX-866 or PI-103. A combination of PI3K and autophagy inhibitors may be a novel strategy for the treatment of T-ALL.

The PI3K/Akt/mTOR pathway participates in the development and progression of acute promyelocytic leukemia, ovarian cancer and other malignant tumors by inhibiting apoptosis, facilitating the cell cycle progression and proliferation, whilst inducing angiogenesis [24, 25]. In particular, the PI3K/Akt/mTOR pathway has been previously reported to be related with the pathogenesis of T lymphocytic leukemia [26]. In the present study, the viability of CCRF-CEM and Jurkat cells was found to be significantly suppressed by PI3K inhibitors (PX-866 and PI-103), which was potentiated further following co-administration with the autophagy inhibitor 3-MA.

Phosphorylation of caspase-9 can be induced to result in the inhibition of the activity of caspase-9 following the activation of AKT, which contributes to the inhibition of the mitochondrial apoptosis pathway [27]. The PI3K/AKT/mTOR pathway has been reported to serve a critical role in the development of leukemia, such that leukemia cell apoptosis can be directly activated by inhibiting this pathway[28]. Therefore, PI3K/AKT/mTOR signaling has become one of the main targets for the treatment of T-ALL [29]. In the present work, after PX-866 and PI-103 treatment, the apoptotic rate of CCRF-CEM and Jurkat cells was significantly elevated. Additionally, the apoptotic rate of T-ALL cells was enhanced further by 3-MA co-treatment. Compared with either PX-866 or PI101 treatment alone, 3-MA co-treatment also dramatically induced the apoptosis of Jurkat cells.

Recently, autophagy has become one of the major mechanisms that can affect the survival of cancer cells,including breast cancer [30]. Meanwhile autophagy provides nutritional support to cells under different stress conditions and is regarded to be one of the physiological processes that can affect cell survival [30]. Autophagy in lung cancer can be inhibited by oncogenes, such as PI3K and mTOR, while it can be activated by autophagy-related proteins, such as ATG5, ATG12 and Beclin-1 [31]. In our work, after the treatment of PX-866 and PI-103, the expression of ATG5, ATG12 and Beclin-1 in CCRF-CEM and Jurkat cells was dramatically elevated. After co-treatment with 3-MA,these autophagy-related proteins were significantly downregulated.

The LC3 protein is closely associated with and is regarded to be a specific biomarker for autophagosome formation [32]. In the present study, the ratio of LC3II/I in CCRF-CEM and Jurkat cells was increased by administration of PX-866 or PI-103. In addition, an increased number of autophagosomes were observed in the PX-866 or PI-103 groups. But this tendency was significantly reversed by 3-MA .

In conclusion, results of our present work suggest that the viability of CCRF-CEM and Jurkat cells was greatly inhibited by the combination of PI3K and autophagy inhibitors, which suppressed autophagy activation induced by the PI3K inhibitor to facilitate the apoptosis of tumor cells.Thus this study provided experimental support for the treatment of T-ALL by demonstrating the suppressive effects of combined PI3K and autophagy inhibitor treatment in this malignancy in vitro.

3-MA, 3-methyladenine; ATG, autophagy-related gene; T-ALL, T-cell acute lymphoblastic leukemia; TEM, transmission electron microscopy.

Funding

The present study was funded by Wenzhou Basic Scientific Research Project (grant no. Y2020747) and the Medical and Health Science and Technology Program of Zhejiang Province (grant no. 2019KY106).

Authors' contributions

XL and WG contributed to study design, statistical analysis, data interpretation and manuscript preparation. YD performed the cell culture, CCK-8 assay, flow cytometry, western blot analysis and manuscript writing. NS and HH conducted the other functional experiments and data collection. XY and MH analyzed of data and searched the literature. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Apoptosis of T-acute lymphoblastic leukemia cells is induced by the combination of PI3K and autophagy inhibitors

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