Physalis alkekengi var. franchetii Extracts Exert Antitumor Effects on Non-Small Cell Lung Cancer and Multiple Myeloma by Inhibiting STAT3 Signaling

Abstract

Background: Physalis alkekengi var. franchetii is an herb that possesses various ethnopharmacological applications. Herein, our current study focuses on the antitumor effect of a combination of physalins, which are regarded as the most representative secondary metabolites from calyces of Physalis alkekengi var. franchetii.

Materials and methods: We mainly investigated the antitumor activity of the physalins extracted from Physalis alkekengi var. franchetii on both solid and hematologic cancers. The main cells used in this study were NCI-H1975 and U266 cells. The major assays used were the CCK-8 assay, Western blot analyses, immunofluorescence assay and Annexin V assay, and a xenograft mouse model was used.

Results: The results showed that physalins exhibited a strong antitumoural effect on both non-small cell lung cancer (NSCLC) and multiple myeloma (MM) cells by suppressing constitutive STAT3 activity and further inhibiting the downstream target gene expression induced by STAT3 signaling, which resulted in the enhanced apoptosis of tumor cells. Moreover, physalins significantly reduced tumor growth in xenograft models of lung cancer.

Conclusion: Collectively, these findings demonstrated that the physalins from Physalis alkekengi var. franchetii may potentially act as cancer preventive or chemotherapeutic agents for NSCLC and MM by inhibiting the STAT3 signaling pathway. The present study served as a promising guide to further explore the precise mechanism of Physalis alkekengi var. franchetii in cancer treatment.

Keywords: Physalis; STAT3; apoptosis; multiple myeloma; non-small cell lung carcinoma.

Figure 1

The main extracts of Physalis alkekengi var. franchetii. (A) Chemical structures of the physalins extracted from Physalis alkekengi var. franchetii. (B) UPLC and total ion current chromatogram. Peaks: 1=physalin A, 2= isophysalin A, 3= physalin O, 4= physalin L and 5= physalin G.

Figure 2

Viability of human tumor cells in the presence of Physalis alkekengi var. franchetii extracts. Cells treated with various doses of physalins (0, 2.5, 5, 10, 15, 20 and 30 μg/mL) for 24 h (A) or 48 h (B). CCK-8 assays were performed to examine cell viability.

Figure 3

Physalis alkekengi var. franchetii extracts suppressed constitutive STAT3 activity, and IL-6-induced STAT3 Tyr705 phosphorylation in NCI-H1975 and U266 cells. (A) NCI-H1975 cells were treated for 4 h with physalins (0, 5, 10, 15 μg/mL). The levels of p-STAT3-Tyr, p-STAT3-Ser, STAT3 and β-actin were detected by Western blot analysis. (B) U266 cells were treated for 4 h with physalins in a dose-dependent manner. The cell lysates were subjected to a Western blot analysis using antibodies specific for p-STAT3-Tyr, p-STAT3-Ser, STAT3 and β-actin. The semiquantification of the protein levels was performed with Image J software. The relative gray values of p-STAT3-Tyr are shown below. (C) Physalins suppressed p-STAT3 nuclear translocation. H1975 cells were treated with 15 μg/mL of physalins for 6 h with or without 25 ng/mL IL-6. Immunofluorescence analysis was performed with an anti-p-STAT3-Tyr primary antibody followed by an anti-rabbit IgG Fab₂ Alexa Fluor 555 antibody. Coverslipped slides were covered with anti-fade reagents with DAPI. The merged images show the overlay of red Alexa Fluor 555 and blue DAPI fluorescence.

Figure 4

Physalis alkekengi var. franchetii extracts suppressed STAT3-mediated downstream genes in NCI-H1975 and U266 cells. (A) NCI-H1975 cells were incubated with various concentrations of the physalins for 24 h. The cell lysates were isolated for Western blot analysis to detect the Bcl-2 family, XIAP and survivin protein levels. β-actin was used as a loading control. (B) U266 cells were treated with physalins in a dose-dependent manner for 24 h. Western blot was performed with the anti-Bcl-2 family, anti-XIAP and anti-survivin primary antibodies. The semiquantification of protein levels was performed with ImageJ software. The relative gray values of Bcl-2 and XIAP are shown below.

Figure 5

Physalis alkekengi var. franchetii extracts induced the poptosis of NCI-H1975 and U266 cells. (A) NCI-H1975 cells and (B) U266 cells were treated with different concentrations of physalins for 24 h, and then, the cells were collected and labeled with Annexin V-FITC/PI followed by flow cytometric analysis. (C) NCI-H1975 cells and (D) U266 cells were treated with physalins in a dose-dependent manner for 24 h. Western blotting was performed with anti-cleaved caspase-9, anti-cleaved caspase-3 and anti-PARP antibodies.

Figure 6

Physalis alkekengi var. franchetii extracts inhibited tumor growth in a human NSCLC xenograft model. (A) Tumor volumes were measured at the indicated times using Vernier calipers. Tumor volume = length × width²/2. ** for P<0.01 and *** for P<0.001. The P values were analyzed by ANOVA with a post hoc test. (B) After 10 days, all the mice were sacrificed, and the tumors were arranged and photographed. (C) The average tumor weights were analyzed in each group. The statistical analysis was performed using Student’s t-test and one-way ANOVA, ** for P<0.01 and *** for P < 0.001. (D) The body weights were measured at the indicated times.