Phloretin attenuates STAT-3 activity and overcomes sorafenib resistance targeting SHP-1-mediated inhibition of STAT3 and Akt/VEGFR2 pathway in hepatocellular carcinoma

Abstract

Background: Hepatocellular carcinoma (HCC) is the most common primary liver malignancy. Phloretin (PH) possesses anticancer, antitumor, and hepatoprotective effects, however, the effects and potential mechanisms of phloretin remain elusive.

Methods: Five HCC cells were tested in vitro for sensitivity to PH, Sorafenib (Sor) or both and the apoptosis, signal transduction and phosphatase activity were analyzed. To validate the role of SHP-1, we used PTP inhibitor III and SHP-1 siRNA. Further, we used purified SHP-1 proteins or HCC cells expressing deletion N-SH2 domain or D61A point mutants to study the PH efficacy on SHP-1. The `in vivo studies were conducted using HepG2 and SK-Hep1 and Sor resistant HepG2SR and Huh7SR xenografts. Molecular docking was done with Swiss dock and Auto Dock Vina.

Results: PH inhibited cell growth and induced apoptosis in all HCC cells by upregulating SHP-1 expression and downregulating STAT3 expression and further inhibited pAKT/pERK signaling. PH activated SHP-1 by disruption of autoinhibition of SHP-1, leading to reduced p-STAT3Tyr705 level. PH induced apoptosis in two Sor-resistant cell lines and overcome STAT3, AKT, MAPK and VEGFR2 dependent Sor resistance in HCCs. PH potently inhibited tumor growth in both Sor-sensitive and Sor-resistant xenografts in vivo by impairing angiogenesis, cell proliferation and inducing apoptosis via targeting the SHP-1/STAT3 signaling pathway.

Conclusion: Our data suggest that PH inhibits STAT3 activity in Sor-sensitive and -resistant HCCs via SHP-1-mediated inhibition of STAT3 and AKT/mTOR/JAK2/VEGFR2 pathway. Our results clearly indicate that PH may be a potent reagent for hepatocellular carcinoma and a noveltargeted therapy for further clinical investigations.

Keywords: Phloretin; SHP-1; STAT3; Sorafenib; hepatocellular carcinoma.

Conflict of interest statement

There are no financial or other interests with regard to this manuscript that might be construed as a conflict of interest. All of the authors are aware of and agree to the content of the manuscript and their being listed as an author on the manuscript.

Figures

Fig. 1

PH inhibits cell proliferation and induces apoptosis in HCCs. a Sor and (b) PH structure. c Cell viability by MTT assay. d DNA fragmentation assay. e Apoptosis was analyzed by annexin V, FACS analysis. Annexin V (+) cells were quantified. f The protein levels of caspase-3 and PARP were determined by Western blot after exposing HCCs to PH for 48 h. Experiments were conducted in triplicate and mean values ± SD (bars) are shown. *p < 0.05, and **p < 0.01 compared to control

Fig. 2

PH inhibits STAT3, AKT/mTOR and RAS/MAPK signaling in HCCs. a Cells were treated for 48h with indicated concentrations of PH and phosphorylated and total protein levels of the target proteins were evaluated by Western blotting. b pSTAT-3 activity was measured by ELISA (c) PH inhibits cytoplasmic, nuclear STAT3 phosphorylation in HCCs at 50uM. d Cells (wild-type or ectopic expression of STAT3) were exposed to PH at 50uM for 24 hours. Percentage of apoptotic cells was analyzed by flow cytometry. e Representative images from two patients with double immunostaing of CD31/SHP1 and pSTAT3. Experiments were conducted in triplicate and mean values ± SD (bars) are shown. *p<0.05, and **p<0.01 compared to control

Fig. 3

PH enhances SHP-1 tyrosine phosphatase activity. a SHP-1 phosphatase activity. b The SHP-1-containing lysates of HCCs were collected by SHP-1 antibody and incubated with PH for 30 min. c PH enhanced SHP-1 activity in cell-free SHP-1 protein. d HCCcells were pre-treated with 25 μM specific SHP-1 inhibitor (PTPIII) for 30 min and then co-incubated with PH 50 μM for additional 48 h. e Knockdown of SHP-1 reversed the biological effects of PH on p-STAT3 and apoptosis. f PH reinforced apoptosis in stably expressing SHP-1 HCCs. g Purified dN1 and D61A mutants of SHP-1 were insensitive to PH treatment. h Percentage of apoptotic cells was analyzed by flow cytometry. i Effect of dN1 and D61A mutants on STAT3 expression. j Molecular model of PH. Experiments were conducted in triplicate and mean values ± SD (bars) are shown. *p < 0.05, and **p < 0.01 compared to control

Fig. 4

PH overcomes Sor resistance in HepG2SR and Huh7SR cells in vitro. a PH inhibited cell growth in Sor resistant HepG2SR and Huh7SR cells. Cell viability was assessed by MTT assay. b PH enhanced DNA fragmentation in SR-HCCs. c The protein levels of caspase-3, caspase-9 and PARP after exposing SR-HCCs to PH. d PH inhibited STAT3, ERK, AKT/mTOR signaling in HCC cells. e pSTAT-3 activity was measured by ELISA. f SHP-1 phosphatase activity in HepG2SR and Huh7SR. g SHP-1 mRNA levels were analyzed by qPCR. h SR-HCC cells were transfected with control siRNA or SHP-1 siRNA for 48 h there exposed to PH and subjected to Western blot assay of p-STAT3 and SHP-1 or seeded on a 6-well plate for the colony forming assay. Experiments were conducted in triplicate and mean values ± SD (bars) are shown. *p < 0.05, and **p < 0.01 compared to control

Fig. 5

PH potentiates the effect of Sor in HepG2SR and Huh7SR cells in vitro. a Effect of PH (50 μM), Sor (10 μM) or both on p-STAT3 and its downstream proteins in SR-HCCs. b SHP-1 phosphatase activity in SR-HCCs exposed to PH, Sor or both for 48 h. c pSTAT3 activity was measured by ELISA. d Colony formation assay. e The protein levels of cleaved caspase-3 and -9 were determined by Western blot assay (f) DNA fragmentation assay for cells exposed to PH, Sor or both for 48 h. Experiments were conducted in triplicate and mean values ± SD (bars) are shown. *p < 0.05, and **p < 0.01 compared to control

Fig. 6

PH inhibits tumor growth via downregulating STAT3-related signaling pathway. a PH significantly inhibited tumor growth in HepG2 and SK-Hep1 tumor-bearing mice. b Tumor weight. c The in vivo SHP-1 phosphatase activity. d The protein levels of p-STAT3 and its downstream proteins were determined by Western blot assay. e STAT3 and related mRNA levels were analyzed by qPCR. f Kaplan–Meier plot showing animal survival after treatment with PH at indicated doses (n = 8). g Immunohistochemical staining (Left panel) of CD31, pSTAT3, cleaved caspase-3, Ki67 and TUNEL in tumor xenografts. Magnification: ×200. Data represents mean values from five random fields per tumor section. Scale bar, 50 μm. Mean values ± SD (bars) are shown. *p < 0.05, and **p < 0.01 compared to control

Fig. 7

PH overcomes the Sor resistance in HepG2SR and Huh7SR xenografts in vivo. a PH significantly reduced tumor growth in SR-HCCs xenografts. When tumors reached 100 mm3, the mice were treated with PH, Sor or both and then tumor growth was measured every 3 days. b Tumor weight was measured on day 30 after tumor excision. c The protein levels of p-STAT3 and its downstream proteins were determined by Western blot. d The in vivo SHP-1 phosphatase activity. e Immunohistochemical staining of CD31, p-STAT3, cleaved caspase-3, Ki67 and TUNEL in HepG2SR and Huh7SR xenografts. Magnification: ×200. Data represents mean values from five random fields per tumor section. Scale bar, 50 μm. Mean values ± SD (bars) are shown. *p < 0.05, and **p < 0.01 compared to control

Fig. 8

Molecular Docking. a PH shows 3 BS of protein with 3H-bond, Sor shows binding near to active site (with 2-H bond). b N-Sh2, C-Sh2, and PTP domains of Shp1 protein (2B3O) structure. The four binding sites (BS) identified from Swiss dock models are marked in the imaged along with the predicted binding affinities for Sor (SFB) and PH (PHL). c Docking of PH in ATP binding site of AKT1 protein (PDB id: 4EJN). PH and 0R4 are in rosy brown and sea green respectively (Left)

Fig. 9

Graphical abstract for the proposed mechanism of action of PH in HCC