Active fraction of ground cherry (Physalis angulata L.) calyces attenuates azoxymethane dextran sulfate sodium‑induced colon carcinogenesis in mice

Abstract

Physalis angulata L., commonly known as wild tomato or ground cherry, is widely used in tropical and subtropical areas to treat health disorders including inflammation, hepatitis, dermatitis, cancer and diabetes. In Colombia, anti-cancer and anti-inflammatory activity are the most common ethnopharmacological applications of P. angulata calyces. P. angulata dichloromethane fraction (PADF) has significant anti-inflammatory activity. The present study assessed the pharmacological effect of PADF on colorectal cancer (CRC) using cancer and normal human cells and an azoxymethane (AOM)/dextran sulfate sodium (DSS) murine model. MTT and clonogenic assay, cell cycle and apoptosis analysis and mitochondrial membrane potential measurement were employed to evaluate in vitro activity of PADF. PADF selectively induced a cytotoxic effect against CRC cells via apoptosis and G2/M arrest. In the AOM/DSS model, treatment with PADF diminished tumor number and size, affected area and expression of proliferating cell nuclear antigen and promoted colon tissue repair. These effects might be related to the increased expression of p38 pro-apoptotic protein in addition to anti-inflammatory activity of PADF demonstrated by decreased levels of TNF-α, IL-6, and IL-1β. PADF may serve as a potential treatment for CRC. Further investigation is warranted to identify the bioactive components in PADF.

Keywords: AOM + DSS; CRC; Physalis angulata L.

Figure 1

PADF affects viability and migration of colorectal cancer cells. (A) Effect of PADF on viability of malignant and normal epithelial colonic cells using MTT assay. (B) Cytotoxic activity of PADF against HT-29 cells spheroids (3D culture). (C) Effect of PADF on colony formation of HT-29 cells. (D) Assessment of PADF influence on HT-29 cell migration. Magnification, x10. n=12. ^**P<0.01, ^***P<0.001 and ^****P<0.0001 vs. untreated PADF, Physalis angulata dichloromethane fraction.

Figure 2

Cell cycle analysis of HT-29 cells following PADF treatment. n=12. ^*P<0.05, ^**P<0.01 and ^****P<0.0001 vs. control. PADF, Physalis angulata dichloromethane fraction; Doxo, Doxorubicin.

Figure 3

Apoptosis induction by PADF in HT-29 cell line. (A) Live, apoptotic and necrotic cell distribution. (B) JC-1 levels following treatment with PADF (6.91-27.64 µg/ml). (C) DNA fragmentation produced by PADF. n=12-32. ^**P<0.01, ^***P<0.001 and ^****P<0.0001 vs. control. PADF, Physalis angulata dichloromethane fraction; MW, molecular weight.

Figure 4

Therapeutic effect of PADF. (A) Experimental design and the body weight change. Tumor (B) number, (C) size and (D) load. (E) Representative macroscopic evaluation of colon tissue. (F) Colon weight/length ratio. n=12-32. ^*P<0.05, ^**P<0.01, ^***P<0.001 and ^****P<0.0001 vs. AOM + DSS. PADF, Physalis angulata dichloromethane fraction; AOM + DSS, azoxymethane + dextran sulfate sodium.

Figure 5

PADF improves microscopic damage induced by AOM + DSS in mice. (A) Representative histopathological examination by hematoxylin-eosin staining (magnification, x8 and x10; black arrow indicates crypt abscess). (B) Histological damage score. (C) Levels of pro-inflammatory cytokines in colonic tissue. (D) Viability of HT-29 cells treated with conditioning media of polarized M1 or M2 RAW 264.7 macrophages exposed to PADF. n=12-32. ^*P<0.05, ^**P<0.01, ^***P<0.001 and ^****P<0.0001 vs. AOM + DSS or untreated. PADF, Physalis angulata dichloromethane fraction; AOM + DSS, Azoxymethane + dextran sulfate sodium.

Figure 6

Expression of signaling proteins implicated in the progression of CRC tumors. (A) Representative western blot analysis of MAPKs (ERK, JNK, and p38) and PCNA in colonic samples. (B) Representative western blot analysis of p38 MAPK, PCNA and β-catenin in HT-29 cells. (C) Quantification of the protein expression of MAPKs (ERK, JNK. and p38) and PCNA in colonic samples. (D) Quantification of protein expression of p38 MAPK, PCNA and β-catenin in HT-29 cells n=4. ^*P<0.05 and ^**P<0.01 vs. AOM + DSS or untreated. PADF, Physalis angulata dichloromethane fraction; AOM + DSS, Azoxymethane + dextran sulfate sodium; PCNA, Proliferating cell nuclear antigen; p-, Phosphorylated.