Pseudolaric Acid B Targets CD147 to Selectively Kill Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) is an aggressive blood cancer. With low survival rates, new drug targets are needed to improve treatment regimens and patient outcomes. Pseudolaric acid B (PAB) is a plant-derived bioactive compound predicted to interact with cluster of differentiation 147 (CD147/BSG). CD147 is a transmembrane glycoprotein overexpressed in various malignancies with suggested roles in regulating cancer cell survival, proliferation, invasion, and apoptosis. However, the detailed function of PAB in AML remains unknown. In this study, AML cell lines and patient-derived cells were used to show that PAB selectively targeted AML (IC50: 1.59 ± 0.47 µM). Moreover, proliferation assays, flow cytometry, and immunoblotting confirmed that PAB targeting of CD147 resulted in AML cell apoptosis. Indeed, the genetic silencing of CD147 significantly suppressed AML cell growth and attenuated PAB activity. Overall, PAB imparts anti-AML activity through transmembrane glycoprotein CD147.


Introduction
Acute myeloid leukemia (AML) is an aggressive hematological malignancy originating in the bone marrow that is characterized by uncontrolled proliferation and accumulation of abnormal blast cells that fail to develop into functional blood cells [1,2].The 5-year net survival rate of 21% among patients aged 15 to 99 years [3] and the dose-limiting toxicities of current regimens [4][5][6][7][8] highlight the glaring need for improved drugs and drug targets.
AC73 is a specific inhibitor of CD147.It has been reported to inhibit CD147 dimerization and prevent migration of hepatocarcinoma cells in vitro and in vivo [33].Mechanistically, it suppresses the ERK/STAT3 activation pathway and imparts cell death by downregulating CD147.However, to significantly inhibit cell proliferation, high doses of AC73 are needed [22,34], which will limit its potential clinical utility.In this study, we evaluated the anti-leukemia effects of PAB, showed that PAB suppressed downstream pathways of CD147 to induce apoptosis of AML cells, and confirmed the role of CD147 in AML proliferation.

PAB Exhibits Anti-Leukemia Activities In Vitro
A drug screen of an in-house library identified PAB as a potent anti-AML compound (Figure 1a).To validate this finding, PAB dose-response curves were generated using a panel of leukemia cell lines.Here, cells were treated with PAB or AC73 at increasing concentrations, and after 72 h, viability was measured using 7-AAD staining (Figure 1b,d).PAB lowered leukemia cell viability at low micromolar concentrations (IC50 value: 1.59 ± 0.47 µM) (Figure 1c).In contrast, the known CD147 inhibitor, AC73, exhibited weak anti-leukemia properties (IC50 value: 32.79 ± 4.18 µM) (Figure 1e).
are needed [22,34], which will limit its potential clinical utility.In this study, we evaluated the anti-leukemia effects of PAB, showed that PAB suppressed downstream pathways of CD147 to induce apoptosis of AML cells, and confirmed the role of CD147 in AML proliferation.

PAB Exhibits Anti-Leukemia Activities In Vitro
A drug screen of an in-house library identified PAB as a potent anti-AML compound (Figure 1a).To validate this finding, PAB dose-response curves were generated using a panel of leukemia cell lines.Here, cells were treated with PAB or AC73 at increasing concentrations, and after 72 h, viability was measured using 7-AAD staining (Figure 1b,d).PAB lowered leukemia cell viability at low micromolar concentrations (IC50 value: 1.59 ± 0.47 µM) (Figure 1c).In contrast, the known CD147 inhibitor, AC73, exhibited weak antileukemia properties (IC50 value: 32.79 ± 4.18 µM) (Figure 1e).
To evaluate selective activity, PAB was tested in normal and patient-derived AML mononuclear cells using colony formation assays.Primary AML colonies were significantly reduced by 60 or 90%, compared to the control, when treated with 1 µM or 2 µM of PAB, respectively (Figure 1f, F2,9 = 54.24,p < 0.0001).In contrast, no significant effects were noted in normal PBSC cells at equivalent concentrations (Figure 1g).These results show that PAB is a potent and selective anti-AML compound.To evaluate selective activity, PAB was tested in normal and patient-derived AML mononuclear cells using colony formation assays.Primary AML colonies were significantly reduced by 60 or 90%, compared to the control, when treated with 1 µM or 2 µM of PAB, respectively (Figure 1f, F 2,9 = 54.24,p < 0.0001).In contrast, no significant effects were noted in normal PBSC cells at equivalent concentrations (Figure 1g).These results show that PAB is a potent and selective anti-AML compound.

PAB Regulates CD147, NF-κB, and Bcl-2 Expression in AML Cell Lines
Chemical proteomics, followed by testing in HeLa cells, showed that PAB targets and reduces CD147 expression [19].To test the effects on CD147 expression, leukemia cells were treated with PAB at doses and times preceding cell death, and protein levels were measured by immunoblotting.PAB, at increasing concentrations, significantly reduced CD147 protein levels in AML cells (Figure 2a,b, F 3,20 = 10.97,p < 0.001; Supplementary Figure S1a,b, F 3,7 = 11.19,p < 0.01).
test was used.

PAB Induces Apoptosis in AML Cells
Overexpression of CD147 is associated with the inhibition of cancer cell apoptosis [23,32,35].Therefore, our observation that PAB reduces CD147 protein levels (Figure 2a,b) led to the hypothesis that PAB decreases CD147 to cause cancer cell apoptosis; a notion supported by several studies [16,18,36].To determine the effects in AML, apoptosis was analyzed by flow cytometry using Annexin-V FITC/7AAD staining (Figure 3a-c).PAB induced AML cell apoptosis, as noted by a significant increase in the ANN + /7AAD -cell population (F 4,42 = 76.98,p < 0.0001).In comparison, AC73 did not induce significant apoptotic effects until reaching 30 µM (F 8,90 = 155.9,p < 0.0001).Indeed, nearly 50 µM of AC73 was required to induce apoptotic effects comparable to that of 1 µM PAB.These findings strongly suggest that PAB can induce apoptosis in AML cells at a relatively low concentration by targeting CD147.A schematic of PAB targeting CD147 is presented in Figure 3d.

PAB Induces Apoptosis in AML Cells
Overexpression of CD147 is associated with the inhibition of cancer cell apoptosis [23,32,35].Therefore, our observation that PAB reduces CD147 protein levels (Figure 2a,b) led to the hypothesis that PAB decreases CD147 to cause cancer cell apoptosis; a notion supported by several studies [16,18,36].To determine the effects in AML, apoptosis was analyzed by flow cytometry using Annexin-V FITC/7AAD staining (Figure 3a-c).PAB induced AML cell apoptosis, as noted by a significant increase in the ANN + /7AAD -cell population (F4,42 = 76.98,p < 0.0001).In comparison, AC73 did not induce significant apoptotic effects until reaching 30 µM (F8,90 = 155.9,p < 0.0001).Indeed, nearly 50 µM of AC73 was required to induce apoptotic effects comparable to that of 1 µM PAB.These findings strongly suggest that PAB can induce apoptosis in AML cells at a relatively low concentration by targeting CD147.A schematic of PAB targeting CD147 is presented in Figure 3d.

Overexpressed in AML Cells, CD147 Regulates AML Cell Growth and Alters NF-κB and Bcl-2 Family Protein Expression
CD147 is overexpressed in cancer cells [25,32], including leukemia cell lines and patient-derived leukemia blasts (as compared to CD34+ hematopoietic progenitor cells) [22].To validate protein level expression, a panel of leukemia cell lines was tested and compared to normal peripheral blood stem cells (PBSCs).CD147 was highly expressed in all AML cell lines tested, while no expression was detected in normal PBSCs (Figure 4a).
Overexpression of CD147 has been linked to cell survival and proliferation in cancer cell lines [32,37].To evaluate the role of CD147 in AML cell proliferation, shRNA-mediated CD147 knockdown (transfected with shRNA-NC or shRNA-CD147-A) was conducted.Knockdown was confirmed by immunoblotting (Figure 4b), and proliferation

Overexpressed in AML Cells, CD147 Regulates AML Cell Growth and Alters NF-κB and Bcl-2 Family Protein Expression
CD147 is overexpressed in cancer cells [25,32], including leukemia cell lines and patient-derived leukemia blasts (as compared to CD34+ hematopoietic progenitor cells) [22].To validate protein level expression, a panel of leukemia cell lines was tested and compared to normal peripheral blood stem cells (PBSCs).CD147 was highly expressed in all AML cell lines tested, while no expression was detected in normal PBSCs (Figure 4a).
Overexpression of CD147 has been linked to cell survival and proliferation in cancer cell lines [32,37].To evaluate the role of CD147 in AML cell proliferation, shRNAmediated CD147 knockdown (transfected with shRNA-NC or shRNA-CD147-A) was conducted.Knockdown was confirmed by immunoblotting (Figure 4b), and proliferation assays showed that leukemia cells lacking CD147 had significantly reduced growth rates (Figure 4c, p < 0.0001).
munoblotting of AML2 cells transfected with shRNA-NC, CD147-A, or CD147-B was conducted.Genetic suppression of CD147 in AML2 cells led to reduced endogenous protein levels of NF-κB (p65), Bcl-2, and Bcl-xL (Figure 4d).There was no change in Bax expression; therefore, lower Bcl-2/Bax and Bcl-xL/Bax ratios were noted (Figure 4e).Together, these results confirm that reductions in CD147 result in cellular effects favoring apoptosis and align with observations from the PAB treatment model.PAB reduced CD147 expression (Figure 2a,b) leading to the suppression of the NF-κB pathway and altered expression of Bcl-2 proteins favoring apoptosis (Figures 2c-g and 3a-c).To determine if CD147 knockdown recapitulates the effects of PAB, the immunoblotting of AML2 cells transfected with shRNA-NC, CD147-A, or CD147-B was conducted.Genetic suppression of CD147 in AML2 cells led to reduced endogenous protein levels of NF-κB (p65), Bcl-2, and Bcl-xL (Figure 4d).There was no change in Bax expression; therefore, lower Bcl-2/Bax and Bcl-xL/Bax ratios were noted (Figure 4e).Together, these results confirm that reductions in CD147 result in cellular effects favoring apoptosis and align with observations from the PAB treatment model.

Knockdown of CD147 Reduces PAB-Mediated Cytotoxicity in AML Cells
To elucidate the role of CD147 in PAB-induced cytotoxicity, AML2 cells were transfected with shRNA-NC or shRNA-CD147-A.Knockdown of CD147 was confirmed by immunoblotting (Figure 5a).The viability of PAB-treated, CD147-knockdown AML2 cells was measured after a 48 h incubation by 7AAD staining.PAB-mediated cytotoxicity was significantly reduced in CD147-knockdown AML2 cells (Figure 5b, p < 0.0001).These findings indicate that CD147 plays a crucial role in mediating the cytotoxic effects of PAB in AML cells.

Knockdown of CD147 Reduces PAB-Mediated Cytotoxicity in AML Cells
To elucidate the role of CD147 in PAB-induced cytotoxicity, AML2 cells were transfected with shRNA-NC or shRNA-CD147-A.Knockdown of CD147 was confirmed by immunoblotting (Figure 5a).The viability of PAB-treated, CD147-knockdown AML2 cells was measured after a 48 h incubation by 7AAD staining.PAB-mediated cytotoxicity was significantly reduced in CD147-knockdown AML2 cells (Figure 5b, p < 0.0001).These findings indicate that CD147 plays a crucial role in mediating the cytotoxic effects of PAB in AML cells.

Discussion
AML is a devastating disease requiring novel therapeutic approaches.In this study, PAB was identified as a molecule capable of inducing selective AML cell toxicity in vitro.Mechanistically, targeting the surface transmembrane protein CD147 resulted in changes in apoptotic proteins favoring leukemia cell death.
CD147 overexpression is associated with cancer cell proliferation and survival, and leukemia cells have elevated levels compared to normal CD34+ hematopoietic progenitor cells [22,25,32].Overexpression of CD147 leads to the dysregulation of the NF-κB pathway in cancer cells [26,30,38], primarily through the degradation of the inhibitor of κB (IκB), which allows for the activation and nuclear translation of p50/p65 dimers [39].Once in the nucleus, active NF-κB dimers can promote the transcription of genes that contribute to the malignant phenotype [40][41][42][43][44]. Specifically, NF-κB can upregulate anti-apoptotic Bcl-2 proteins (e.g., Bcl-2 and Bcl-xL) [31,[45][46][47], which in turn suppress pro-apoptotic proteins (e.g., Bax and Bak), thereby promoting cell survival and proliferation [48][49][50].Consequently, the inhibition of CD147 suppresses the NF-κB pathway and alters anti-and proapoptotic protein ratios to favor apoptosis induction.In this study and consistent with previous observations, CD147 was highly expressed in AML but not in normal PBSCs.This differential expression confirms that CD147 could serve as a unique molecular target

Discussion
AML is a devastating disease requiring novel therapeutic approaches.In this study, PAB was identified as a molecule capable of inducing selective AML cell toxicity in vitro.Mechanistically, targeting the surface transmembrane protein CD147 resulted in changes in apoptotic proteins favoring leukemia cell death.
CD147 overexpression is associated with cancer cell proliferation and survival, and leukemia cells have elevated levels compared to normal CD34+ hematopoietic progenitor cells [22,25,32].Overexpression of CD147 leads to the dysregulation of the NF-κB pathway in cancer cells [26,30,38], primarily through the degradation of the inhibitor of κB (IκB), which allows for the activation and nuclear translation of p50/p65 dimers [39].Once in the nucleus, active NF-κB dimers can promote the transcription of genes that contribute to the malignant phenotype [40][41][42][43][44]. Specifically, NF-κB can upregulate anti-apoptotic Bcl-2 proteins (e.g., Bcl-2 and Bcl-xL) [31,[45][46][47], which in turn suppress pro-apoptotic proteins (e.g., Bax and Bak), thereby promoting cell survival and proliferation [48][49][50].Consequently, the inhibition of CD147 suppresses the NF-κB pathway and alters anti-and pro-apoptotic protein ratios to favor apoptosis induction.In this study and consistent with previous observations, CD147 was highly expressed in AML but not in normal PBSCs.This differential expression confirms that CD147 could serve as a unique molecular target in AML.Further, the knockdown of CD147 in AML cells suppressed growth and proliferation, reduced protein levels of NF-κB (p65), Bcl-2, Bcl-xL, and lowered ratios of Bcl-2/Bax and Bcl-xL/Bax.These findings confirm the link between CD147 and the regulation of downstream targets (i.e., NF-κB and Bcl-2 family proteins) in AML, suggesting the role of CD147 in signaling pathways that contribute to disease pathophysiology.
PAB had a low IC50 (1.59 ± 0.47 µM) and induced apoptosis by targeting CD147.Indeed, PAB-treated cells had a phenotype resembling CD147 knockdown cells.The pharmacological inhibition, or genetic knockdown, of CD147 reduced AML cell growth and decreased levels of NF-kB and ratios of Bcl-2/Bax and Bcl-xL/Bax.Finally, leukemia cells with decreased CD147 expression were less sensitive to PAB, further supporting the role of CD147 in PAB-induced death.While these results point toward a unique mechanism of PAB-induced selective AML cell apoptosis, the detailed molecular mechanism by which PAB targets CD147 requires further investigation.In addition, the bioavailability of PAB, which would significantly impact its therapeutic potential and optimal dosing strategies, requires characterization.Finally, the absence of in vivo studies limits our understanding of PAB's pharmacodynamics and is a focus of current research.
In summary, this study demonstrated the potent in vitro anti-leukemia activity of PAB, a plant-derived bioactive compound, against AML by specifically targeting and inhibiting CD147, a transmembrane protein of the immunoglobulin superfamily, leading to the suppression of CD147's downstream anti-apoptotic pathways and induction of apoptosis in AML cells.
Cell lines were grown in T25 or T75 vented filter cap tissue culture flasks (Sarstedt; Nümbrecht, Germany) and incubated in 5% CO 2 at 37 • C.

Cell Viability Assay
Cell viability was measured using the 7-aminoactinomycin D exclusion assay (7-AAD; Cayman Chemicals).Leukemia cells were seeded in triplicate on a 96-well plate at a concentration of 1.25-5 × 10 5 cells/mL (in 95 µL fresh media).Cells were treated with increasing concentrations of PAB or AC73 (5 µL of each drug to produce the desired final concentration).The remaining wells were filled with 100 µL of phosphate-buffer saline (PBS).After 48 or 72 h, cells were harvested by centrifugation at 1200 rpm for 5 min, resuspended in 250 µL of 1 µg/mL 7-AAD, diluted in PBS and analyzed using a flow cytometer (Guava EasyCyte 8HT; EMD Millipore; Burlington, MA, USA), as previously described [59].

Apoptosis Assay
Cell viability and apoptosis were measured using Annexin-V FITC/7-AAD exclusion.Leukemia cells were seeded in 96-well plates (1.25-5 × 10 5 cells/mL) and treated with increasing doses of PAB or AC73 for 24 h.Following treatment, cells were collected and resuspended in 50 µL of staining solution consisting of an Annexin V binding buffer (Biovision; Exton, PA, USA) with Annexin V-FITC (150 µg/mL; Biovision) and incubated in the dark for 15 min at room temperature.The cells were then washed with 100 µL Annexin V binding buffer, centrifuged at 1200 rpm for 5 min, and incubated with 7AAD (1 µg/mL; Cayman Chemicals) for 5 min.Following incubation, fluorescence was detected by flow cytometry (Guava ® easyCyteTM 8HT; EMD Millipore) as previously described [60].

Colony Formation Assays
Colony formation assays were performed with primary cells obtained with written informed consent provided by Princess Margaret Cancer Centre (Toronto, ON, Canada).Cells were stored in 10%DMSO, 50% FCS, and alpha-MEM at −150 • C. Primary cells (i.e., patient-derived human AML mononuclear cells or normal peripheral blood stem cells (PBSCs) from healthy donors) were collected according to approved human ethics protocols.Cells were suspended in MyeloCult TM H5100 media (StemCell Technologies; Vancouver, BC, Canada).In a 15 mL tube, 300 µL of cell suspension was added to 3 mL of MethoCult GF H4435-enriched methylcellulose medium (StemCell Technologies).The cells were plated in a 35 mm cell culture dish (Corning; Tewksbury, MA, USA) with 1 × 10 5 cells/dish for patient-derived AML cells or 1 × 10 4 cells/dish for normal PBSCs using a 5 mL syringe with a blunt cannula (Covodein; Minneapolis, MN, USA); increasing doses of PAB (0, 1, and 2 µM) were added.Duplicates of each treatment were incubated in 100 mm cell culture dishes (Corning) with an additional uncapped 35 mm dish containing distilled water to control the humidity.The plates were incubated for 7-14 days at 37 • C with 5% CO 2 and 95% relative humidity.The colonies were counted on an inverted microscope; clusters of 50 or more cells were counted as one colony.

Retroviral Mediated Knockdown of CD147
CD147 gene-specific short hairpin RNA (shRNA-CD147-A and shRNA-CD147-B; OriGene; Rockville, MD, USA; see Supplementary Table S1 for sequences) and a scrambled shRNA negative control (shRNA-NC; OriGene) were transfected into HEK293T cells (cultured in antibiotic-free DMEM media with 10% FBS).Transfection was carried out with a TransIT-LT1 transfection reagent (MirusBio; Madison, WI, USA), according to the manufacturer's protocol.The cells were cultured at 37 • C after transfection.At 48-72 h post-transfection, the media was collected from the culture and centrifuged at 2000× g for 5 min to remove the cell debris.The supernatant was used as viral stock for further transduction.

Figure 1 .
Figure 1.PAB is a potent anti-leukemia compound.(a) A drug screen identified PAB as a potent anti-AML drug compound.(a, Top) PAB and AC73 structure.Leukemia cell lines were treated with increasing concentrations of (b) PAB or (d) AC73.After 72 h, cell viability was measured by flow

Figure 1 .
Figure 1.PAB is a potent anti-leukemia compound.(a) A drug screen identified PAB as a potent anti-AML drug compound.(a, Top) PAB and AC73 structure.Leukemia cell lines were treated with increasing concentrations of (b) PAB or (d) AC73.After 72 h, cell viability was measured by flow cytometry using 7-AAD staining.IC50 values for (c) PAB and (e) AC73 were calculated from the dose responses in (b,d).Values were the average of three replicates performed in triplicate (n = 3).(f) Primary patient-derived human AML mononuclear cells were incubated with 1 or 2 µM PAB, and colonies were counted after 14 days (n = 4).(g) Peripheral blood stem cells (PBSCs) from healthy donors were incubated with 1 or 2 µM PAB, and colonies were counted after 14 days (n = 4).*** p ≤ 0.001, **** p ≤ 0.0001.Unpaired, one-way ANOVA paired with a Dunnett's multiple comparisons test was used.

Figure 4 .
Figure 4. CD147 is highly expressed in leukemia cells but not in normal peripheral blood stem cells (PBSCs) and knockdown impairs growth.(a) Immunoblotting for CD147 protein levels in leukemia cell lines and normal PBSCs.(b) AML2 cells were transfected with scrambled shRNA (negative control, NC) or shRNA-CD147-A (i.e., targeting CD147).CD147 knockdown was validated by