Palmatine suppresses acute myeloid leukemia (AML) by inducing pyroptosis via ROS/PI3K/AKT signaling

doi:10.21203/rs.3.rs-1739045/v1

Purpose Palmatine (PAL) is widely identified as an isoquinoline alkaloid among the representatives of the Berberidaceae botanical family. Palmatine exerts suppressive effects on several kinds of cancer. Several natural products were found to suppress acute myeloid leukemia (AML); however, the effects of Palmatine on AML are still unknown. In this study, we aim to evaluate the effects of PAL on AML.

Methods Several AML cell lines, including HL-60, OCI-AML3, Molm-13 and KG-1 cells were employed to evaluate the effects of PAL. Cell proliferation, migration, invasion, tumor formation in soft agar were evaluated. PAL’s effects on ROS accumulation and subsequent induction of pyroptosis were measured.

Results PAL promoted the cleavage of caspase-3 and PARP1 and induced apoptosis. PAL treatment also caused the release of lactic dehydrogenase (LDH) and the depolarization of the mitochondrial membrane potential and subsequently induced the pyroptosis-specific cleavage of caspase-1, caspase-5 and gasdermin D, which was reversed by pyroptosis inhibitor VX765. After PAL treatment for 6-24 hours, PAL treatment increased ROS in HL-60 and KG-1 cells. Abolishment of ROS with N-acetyl cysteine (NAC) attenuated LDH release, which was similar to the effect of VX765, indicating that palmatine contributes to the suppression of AML partially by inducing pyroptosis. Furthermore, we found that palmatine-induced ROS accumulation inhibited PI3K and AKT phosphorylation and abolished ROS-mediated reactivation of PI3K/AKT signaling, which was similar to the effects of the PI3K activator 740Y-P.

Conclusion Taken together, these results indicate that palmatine induces cell death mainly by inducing ROS accumulation and ROS/PI3K/AKT signaling.

Palmatine

pyroptosis

reactive oxygen species (ROS)

acute myeloid leukemia (AML)

lactic dehydrogenase (LDH)

Palmatine (PAL), a bright yellow compound, is a natural isoquinoline alkaloid. It is structurally similar to berberine, but the two differ slightly with respect to their substitution of the isoquinoline structure. PAL exhibits a wide spectrum of pharmacological and biological properties, including anti-inflammatory, antiviral, and neuroprotective activities (Ali 2013), and has been used to treat jaundice, dysentery, hypertension, inflammation and liver-related diseases (Fan 2020). Moreover, PAL and its derivatives have also been shown to exhibit significant antitumor activity in a variety of cells, such as prostate cells (DU145) (Hambright 2015), hepatocytes (QGY-7701, SMMC-7721 and HepG2), human breast cancer cells (MCF-7), malignant glioblastoma cells (U251), colon cancer cells (HT-29) and cervical cancer cells (SiHa) (Long 2019).

Pyroptosis, also known as cellular inflammatory necrosis, is a kind of programmed cell death mediated by gasdermin. It is manifested by cell swelling until the cell membrane ruptures, resulting in the release of cell contents and activation of a strong inflammatory response. Pyroptosis is closely related to innate immunity and the disease process. Evidence is mounting that pyroptosis can affect tumor progression and may be closely associated with various tumors, such as melanoma (Yu 2019) ^[5], breast cancer (Kolb 2016; Jiao 2020), colorectal cancer (Yu 2019), gastric cancer (Wang 2018), liver cancer (Chu 2016), cervical cancer (Bostanci 2017), lung cancer (Zhang 2019), and leukemia (Johnson 20187). Studies have shown that the pyroptosis of a small number of tumor cells can effectively regulate the tumor immune microenvironment and activate a strong T-cell-mediated antitumor immune response (Wang 2020). With this discovery, new strategies can be applied for the development of tumor immunotherapy drugs. It was also found that chemotherapy drugs induce caspase-3 activation and pyroptosis in tumor cells with high GSDME expression. However, in tumor cell lines with low expression of GSDME, decitabine may upregulate the expression of GSDME and increase the sensitivity of tumor cells to chemotherapeutic drugs, thereby making these cells more susceptible to pyroptosis (Wang 2017; Rogers 2017).

In tumor cells, antioxidant proteins are highly expressed to detoxify elevated ROS levels, and redox homeostasis is established to maintain precarcinogenic signals and resistance to apoptosis. Compared with normal cells, tumor cells have an altered redox balance and are in a relatively high redox state, suggesting that ROS manipulation is a potential strategy for cancer therapy. Disrupted mitochondrial membrane potential (MMP) and ROS generation are often associated with pyroptosis. Pyroptosis is mediated by GSDMD, while the breakdown of MMP is generated by ROS induced by NLRP3. The elimination of ROS alleviates the cleavage of Gsdmd, suggesting that Gsdmd cleavage occurs downstream of ROS release. Consistent with this result, hydrogen peroxide can promote the cleavage of Gsdmd through caspase-1. In fact, four amino acid residues of Gsdmd are oxidized under oxidative stress. The efficiency of Gsdmd cleavage by inflammatory caspase-1 is dramatically reduced when oxidative modification is blocked by mutation of these amino acid residues. These results indicate that Gsdmd oxidation serves as a new mechanism by which mitochondrial ROS promote NLRP3 inflammation-dependent pyroptosis.

In this study, we investigated the antitumor effect of PAL on AML and clarified its effect on the mitochondrial activity of AML cells from the perspective of its effect on ROS and the PI3K/AKT signaling pathway to investigate its mechanism of action in malignant tumors.

Cell lines

The human AML cell lines HL-60, OCI-AML3, KG-1, and Molm-13 were acquired from American Type Culture Collection (Rockville, MD, USA). HL-60 and OCI-AML3 cells were maintained in DMEM (Gibco, Life Technologies, Carlsbad, CA, USA) supplemented with 20% fetal bovine serum (FBS) (Gibco), 1% penicillin–streptomycin (Gibco) and 4 mM l-glutamine (Gibco). KG-1 and Molm-13 cells were cultured using RPMI-1640 medium (Gibco) supplemented with 10% FBS, 1% penicillin–streptomycin (Gibco) and 2 mM l-glutamine (Gibco). Cells were grown at 37 °C in 5% CO₂. Every three days, half of the medium was replaced.

CCK-8 Assay

The Cell Counting Kit-8 (CCK-8) assay was employed to analyze the effects of PAL on cell proliferation. The CCK-8 assay was performed using 2 × 10⁴ cells/well in 96-well plates. After the cells were cultured with 2.5, 5, 7.5 or 10 μg/mL PAL, CCK-8 (10 μL) was added for an additional 4-hour incubation at 37°C. Cell suspensions were then prepared and vortexed for 5 min, after which the absorbance was read at 450 nm, and the cell proliferation rate was determined.

Apoptosis assay

Annexin V-FITC antibody immunofluorescence combined with PI/DNA binding was adopted for fluorescent analysis of apoptosis. Cells (1× 10⁶) treated with 5 μg/mL PAL for 24 h were collected and subjected to quantitative flow cytometry analysis according to the instructions of the Annexin V-FITC kit (BioVision, USA).

Western blot analysis

Cells (1 × 10⁶) were collected and washed three times with PBS. Total protein was extracted by using an Animal Tissue/cells/bacteria total protein isolation kit (DocSense, Chengdu, China) according to the manufacturer’s instructions. The protein concentration was then determined by using a BCA protein kit (Sigma–Aldrich, St. Louis, MO, USA). Twenty micrograms of total protein was separated by SDS–PAGE and electrophoretically transferred onto a PVDF membrane (Millipore, Billerica, MA, U.S.A.). The membrane was blocked with 5% nonfat milk in TBST buffer and incubated overnight at 4 °C with specific primary antibodies, including anti-cleaved caspase-3 (cat. No.: ab32042), anti-cleaved PARP1 (cat. No.: ab32064), anti-beta actin (cat.:: ab8226), anti-pro-caspase-1 (cat. No.: 2225T, CST), anti-cleaved caspase-1 (cat. No.: 24232S, CST), anti-pro-caspase-5 (cat. No.: ab40887, Abcam), anti-cleaved caspase-5 (cat. No. LS-C380468-50, LSBio), anti-GSDMD (cat. No.: ab219800, Abcam), anti-cleaved GSDMD (cat. No.: 10137, CST) antibodies. The membrane was washed with TBST buffer and incubated with the appropriate secondary antibody (horseradish peroxidase-conjugated goat-anti-mouse IgG). Analysis was performed using enhanced chemiluminescence kits (Millipore, Billerica, MA, USA).

Measurement of intracellular ROS.

Cells were exposed to PAL alone or in combination with NAC or VX765 for the indicated time points (6, 12, 18, 24 h), the supernatants were removed, and the cells were labeled with 2′,7′-dichlorofluorescin diacetate (DCFH-DA, Invitrogen, Paisley, UK) at 37°C for 30 min. The cells were washed twice with PBS and maintained in 1 ml serum-free medium. Cellular ROS levels were analyzed using a fluorescent enzyme labeling instrument (Spectra Max M5; Molecular Devices LLC).

JC-1 staining

The mitochondrial membrane potential was detected using a JC-1 mitochondrial membrane potential assay kit (cat. No.: C2006; Beyotime Institute of Biotechnology). Briefly, the cells were collected and washed three times with PBS and then incubated with JC-1 staining solution for 20 min at 37°C. The cells were then washed twice with 1x JC-1 buffer, and the fluorescence intensity was analyzed using flow cytometry.

Colony formation in soft agar

To assess tumor formation ability in vitro, soft agar clonogenic assays were performed. Each well of a 6-well plate contained 2 mL of 0.5% (w/v) low-melting agar (Sigma–Aldrich, St. Louis, MO, USA) in DMEM with 10% FBS. The cells were mixed equally, and 1×10⁴ cells in 2 mL of 0.3% low-melting agar in 10% FBS were added above the polymerized base solution. The plates were incubated (37°C, 5% CO₂) for 14 days before colony number and diameter were quantified microscopically.

Cell migration and invasion assay

Transwell migration assay experiments were performed using a 24-well Transwell chemotaxis chamber technique (Millipore, Billerica, MA, USA). Briefly, DMEM/F12 (500 μL) supplemented with 10% FBS was placed in the lower chamber. A total of 1× 10⁴ cells in 200 μL medium were seeded into the upper chamber (pore size, 8 μm). The chamber was then incubated for 24 h at 37 °C in a humidified atmosphere with 5% CO₂. The membrane (Millipore) was removed, and its upper surface was wiped away with a cotton swab to remove the unmigrated cells. The membrane was then merged in 4% paraformaldehyde for 5 min at room temperature and stained with 0.1% crystal violet for 10 min followed by 3 washes with ice-cold PBS. The number of cells that migrated to the lower surface of the membrane was counted in 10 random high-power fields (HPFs) under a light microscope (BL-AC10DS, Olympus, Tokyo, Japan). Each assay was performed in triplicate wells.

To analyze invasion, the underlying surface of the membrane was coated with matrix gel (0.01%) at 37°C for 2 h. The lower chamber was filled with 1 ml of DMEM supplemented with 10% FBS. A total of 1×10⁴ cells in a volume of 0.2 ml were added to the upper chamber. After incubation at 37°C for 24 h, the cells on the upper surface of the Transwell membrane were removed. The cells attached to the lower surface of the membrane were stained with 1% crystal violet and imaged.

Statistics and presentation of data

The results are expressed as the mean±SD. Statistical analysis involving two groups was performed using Student’s t test. All data were processed with SPSS 19 version software.

In this study, we evaluated the suppressing roles of PAL in AML cells. PAL induces both apoptotic and pyroptotic cell death in AML cells by inducing ROS accumulation and thus inhibiting PI3K/AKT signaling. Moreover, PAL treatment expectedly disturbed mitochondrial homeostasis by inducing ROS, which is consistent with the hallmarks of pyroptosis.

Pyroptosis is a newly discovered model of proinflammatory cell death that is triggered by different inflammation-related caspases. Multiple inflammasome complexes are assembled and activated under the stimulation of intracellular and extracellular pathological signals, leading to the activation of inflammatory caspases. First, activated caspase cleaves the precursors of inflammatory factors (IL-1β and IL-18) to promote their maturation; second, it activates and cleaves GSDMD, resulting in cell membrane pore formation and finally cell permeablization, cell content release and pyroptosis (Kayagaki 2015; Shi 2015; Broz 2016; Rogers 2019). In addition, mitochondrial apoptosis can induce the activity of caspase-1 mediated by NLRP3 inflammatory bodies (Tsuchiya 2019), which is largely dependent on the activity of potassium channel glycoprotein mediated by caspase-3. After PAL treatment, without affecting caspase-1 or gasdermin D, cleaved caspase-1 and cleaved gasdermin D levels were significantly increased. Although LDH release is widely observed in both apoptotic (Hu 2022) and pyroptotic cells (Xu 2021), the disturbance of mitochondrial homeostasis was still observed after PAL treatment. These results demonstrated that PAL treatment stimulated both apoptotic and pyroptotic cell death in AML cells.

The PI3K/AKT pathway is involved in many key cellular functions, including protein synthesis, cell cycle progression, proliferation, apoptosis, autophagy, and drug resistance, in response to the stimulation of growth factors (EGF, PDGF, NGF and VEGF), hormones (PGE) and cytokines (IL-17, IL-6 and IL-2) (Qiu 2014). PAL treatment slightly affected AKT or PI3K total protein levels and decreased the phosphorylation of PI3K and AKT, which was reversed by the PI3K activator or the ROS scavenger NAC. These results indicated that PAL treatment regulates PI3K/AKT signaling by inducing ROS accumulation. Activation of PI3K/AKT signaling or scavenging of ROS by added NAC also reversed the increase in LDH release and the decrease in cell viability. These results demonstrated that PAL treatment induces pyroptosis mainly by regulating PI3K/AKT signaling.

ROS, on the one hand, directly activate PI3K to amplify its downstream signal transduction and, on the other hand, inactivate phosphatase and tensin homolog (PTEN). It inhibits PIP3 synthesis through oxidized cysteine residues in the active center, thereby inhibiting the activation of Akt (Ahn 2012). Furthermore, ROS can promote the phosphorylation of PTEN through casein kinase II, thereby promoting the degradation of PTEN via the protein pathway (Kim 2008). Meanwhile, protein phosphatase 2A (PP2A), which may be inactivated by ROS, can inhibit Akt/PKB. Nevertheless, it appears that ROS oxidize the disulfide bridge in Akt/PKB at lower levels. As a result, Akt/PKB is associated with PP2A for short-term activation of Akt/PKB (Lee 2002; Murata 2003; Zhou 2018). As a limitation, we failed to evaluate whether PAL treatment regulates mitochondrial ROS, which is worth further investigation.

PAL treatment induces ROS accumulation, mitochondrial dysfunction, LDH release and cell death, which is like the effects of carbonyl cyanide m-chlorophenyl hydrazone (CCCP), which activates oxidative stress. In melanoma A375 cells, oxidative stress may promote Bax supplementation in mitochondria, causing cytochrome C to be released into the cytoplasm (Chang 2021). In this way, caspase-3 is activated, which ultimately leads to the cleavage of GSDME and pyroptosis. Based on this, it can be concluded that iron can enhance the sensitivity of drugs that activate ROS in vivo and amplify the signal of ROS to drive pyroptosis [34], revealing a potential iron-based intervention strategy for the treatment of melanoma patients.

We also evaluated the effects of 2.5 μg/mL PAL on malignant behaviors, including tumor formation, migration and invasion in AML cells. As expected, all these behaviors were suppressed by PAL treatment. Although we failed to detect the exact mechanisms of these inhibitory roles of PAL, further investigation is warranted.

In summary, we revealed a detailed mechanism by which PAL restrains cell growth, tumor formation, migration, and invasion and induces both apoptotic and pyroptotic cell death by inducing ROS accumulation and thus regulating PI3K/AKT signaling in AML. The PAL/ROS/PI3K/AKT regulatory axis might be a promising therapeutic target and contribute to the clinical treatment of AML.

In this study, we evaluated the suppressing roles of PAL in AML cells. PAL induces both apoptotic and pyroptotic cell death in AML cells by inducing ROS accumulation and thus inhibiting PI3K/AKT signaling. Moreover, PAL treatment expectedly disturbed mitochondrial homeostasis by inducing ROS, which is consistent with the hallmarks of pyroptosis.

Pyroptosis is a newly discovered model of proinflammatory cell death that is triggered by different inflammation-related caspases. Multiple inflammasome complexes are assembled and activated under the stimulation of intracellular and extracellular pathological signals, leading to the activation of inflammatory caspases. First, activated caspase cleaves the precursors of inflammatory factors (IL-1β and IL-18) to promote their maturation; second, it activates and cleaves GSDMD, resulting in cell membrane pore formation and finally cell permeablization, cell content release and pyroptosis (Kayagaki 2015; Shi 2015; Broz 2016; Rogers 2019). In addition, mitochondrial apoptosis can induce the activity of caspase-1 mediated by NLRP3 inflammatory bodies (Tsuchiya 2019), which is largely dependent on the activity of potassium channel glycoprotein mediated by caspase-3. After PAL treatment, without affecting caspase-1 or gasdermin D, cleaved caspase-1 and cleaved gasdermin D levels were significantly increased. Although LDH release is widely observed in both apoptotic (Hu 2022) and pyroptotic cells (Xu 2021), the disturbance of mitochondrial homeostasis was still observed after PAL treatment. These results demonstrated that PAL treatment stimulated both apoptotic and pyroptotic cell death in AML cells.

The PI3K/AKT pathway is involved in many key cellular functions, including protein synthesis, cell cycle progression, proliferation, apoptosis, autophagy, and drug resistance, in response to the stimulation of growth factors (EGF, PDGF, NGF and VEGF), hormones (PGE) and cytokines (IL-17, IL-6 and IL-2) (Qiu 2014). PAL treatment slightly affected AKT or PI3K total protein levels and decreased the phosphorylation of PI3K and AKT, which was reversed by the PI3K activator or the ROS scavenger NAC. These results indicated that PAL treatment regulates PI3K/AKT signaling by inducing ROS accumulation. Activation of PI3K/AKT signaling or scavenging of ROS by added NAC also reversed the increase in LDH release and the decrease in cell viability. These results demonstrated that PAL treatment induces pyroptosis mainly by regulating PI3K/AKT signaling.

ROS, on the one hand, directly activate PI3K to amplify its downstream signal transduction and, on the other hand, inactivate phosphatase and tensin homolog (PTEN). It inhibits PIP3 synthesis through oxidized cysteine residues in the active center, thereby inhibiting the activation of Akt (Ahn 2012). Furthermore, ROS can promote the phosphorylation of PTEN through casein kinase II, thereby promoting the degradation of PTEN via the protein pathway (Kim 2008). Meanwhile, protein phosphatase 2A (PP2A), which may be inactivated by ROS, can inhibit Akt/PKB. Nevertheless, it appears that ROS oxidize the disulfide bridge in Akt/PKB at lower levels. As a result, Akt/PKB is associated with PP2A for short-term activation of Akt/PKB (Lee 2002; Murata 2003; Zhou 2018). As a limitation, we failed to evaluate whether PAL treatment regulates mitochondrial ROS, which is worth further investigation.

PAL treatment induces ROS accumulation, mitochondrial dysfunction, LDH release and cell death, which is like the effects of carbonyl cyanide m-chlorophenyl hydrazone (CCCP), which activates oxidative stress. In melanoma A375 cells, oxidative stress may promote Bax supplementation in mitochondria, causing cytochrome C to be released into the cytoplasm (Chang 2021). In this way, caspase-3 is activated, which ultimately leads to the cleavage of GSDME and pyroptosis. Based on this, it can be concluded that iron can enhance the sensitivity of drugs that activate ROS in vivo and amplify the signal of ROS to drive pyroptosis [34], revealing a potential iron-based intervention strategy for the treatment of melanoma patients.

We also evaluated the effects of 2.5 µg/mL PAL on malignant behaviors, including tumor formation, migration and invasion in AML cells. As expected, all these behaviors were suppressed by PAL treatment. Although we failed to detect the exact mechanisms of these inhibitory roles of PAL, further investigation is warranted.

In summary, we revealed a detailed mechanism by which PAL restrains cell growth, tumor formation, migration, and invasion and induces both apoptotic and pyroptotic cell death by inducing ROS accumulation and thus regulating PI3K/AKT signaling in AML. The PAL/ROS/PI3K/AKT regulatory axis might be a promising therapeutic target and contribute to the clinical treatment of AML.

Acknowledgements

The author would like to thanks for Mr Tao Hong for her language editing and her suggestion of statistical analysis.

Funding

This work was supported by The General Program (Key Program, Major Research Plan) of National Natural Science Foundation of China http://isisn.nsfc.gov.cn/egrantweb/ (No. 82074298), the Technical Innovation Research and Development Project of Scientific and Technological Office of Chengdu (2021-YF05-01726-SN) and Med-X Centre for Informatics funding project of Sichuan University ( No. YGJC014)

Competing Interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Authors' contributions

QZ, ZYZ and QYH contributed to experimental design. QZ, YQP and YCL are responsible for performing cell culture related experiments. QZ and HQC collected data and performed statistical analysis. QZ and ZYZ wrote manuscript and make modifications. All authors read manuscript.

Data Availability

All data is included in this manuscript

Ethics approval and consent to participate

No applicated

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No competing interests reported.

Palmatine suppresses acute myeloid leukemia (AML) by inducing pyroptosis via ROS/PI3K/AKT signaling

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Abstract

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Introduction

Materials And Methods

Results

Discussion

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Additional Declarations

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