Evaluation of anti-tumorigenic activity of BP3B against colon cancer with patient-derived tumor xenograft model

Abstract

Background: KIOM-CRC#BP3B (BP3B) is a novel herbal prescription that is composed of three plant extracts. Our preliminary study identified that BP3B exhibited potent anti-proliferative activity against various types of cancer cell lines in vitro. Because the in vivo anti-tumor effect of BP3B is not evaluated before clinical trial, we want to test it using patient's samples.

Methods: To confirm the in vivo anti-cancer effect of BP3B, we used genetically characterized patient-derived colon tumor xenograft (PDTX) mouse model. Anti-cancer activity was evaluated with apoptosis, proliferation, angiogenesis and histological analysis.

Results: Oral administration of BP3B significantly inhibited the tumor growth in two PDTX models. Furthermore, TUNEL assay showed that BP3B induced apoptosis of tumor tissues, which was associated with degradation of PARP and Caspase 8 and activation of Caspase 3. We also observed that BP3B inhibited cancer cell proliferation by down-regulation of Cyclin D1 and induction of p27 proteins. Inhibition of angiogenesis in BP3B-treated group was observed with immunofluorescence staining using CD31 and Tie-2 antibodies.

Conclusion: These findings indicated that BP3B has a strong growth-inhibitory activity against colon cancer in in vivo model and will be a good therapeutic candidate for treatment of refractory colon cancer.

Keywords: BP3B; Colon cancer; Medicinal plant; Patient-derived tumor xenograft.

Fig. 1

The inhibitory effect of BP3B on in vivo tumor growth in PDTX model. Patient-derived colon tumors were subcutaneously established in BALB/c nude mice. Representative PDTX samples were resected on day 21 (five tumors per group) showing the difference in tumor volumes between vehicle (0.5% Na-CMC), extracts (250 and 500 mg/kg) and oxaliplatin (5 mg/kg) (a). When the tumors reached 100-200 mm³ in size, mice were treated with drugs for 3 weeks. Tumor sizes were measured every 3 days using a caliper and tumor volumes were calculated (b) and body weight (c). ** p < 0.01 and *** p < 0.001

Fig. 2

Histological analysis of tumor samples after BP3B administration. After sacrificing the mice, colon tumor tissues from vehicle (a and e), oxaliplatin (5 mg/kg, b and f), low dose (250 mg/kg, c and g) and high dose (500 mg/kg, d and h)-treated group were fixed and checked with hematoxylin/eosin-staining. Cell nuclei were stained with hematoxylin (purple). Red arrows indicate the representative cells with apoptotic characteristics and cell death. Scale bars are 50 μm

Fig. 3

Induction of tumor apoptosis by BP3B in PDTX model. a To measure the apoptotic tissues in BP3B-treated tumors, TUNEL assay was performed. Apoptotic cells were visualized into green and nuclei were stained with DAPI (blue). The intensity of image was calculated with ImmunoRatio software. Scale bars are 50 μm. ** p < 0.01 and *** p < 0.001. b Expression pattern of apoptosis-related proteins (PARP and Cleaved-caspase3) was confirmed with western blot after lysis of tumor tissues. Densitometric analysis was performed with Image J software

Fig. 4

Inhibition of cell proliferation by BP3B in PDTX model. a To check the effect of BP3B on cell proliferation, immunohistochemical analysis of Ki67 was performed with drug-treated samples. Ki67 were stained into brown and nuclei were counterstained with hematoxylin (purple). The intensity of Ki67-positive cell was calculated with ImmunoRatio software. Scale bars are 50 μm. * p < 0.05, ** p < 0.01, *** p < 0.001. b Expression of cell proliferation markers (Cyclin D1 and p27) was confirmed with western blot. Densitometric analysis was performed with Image J software

Fig. 5

Suppression of angiogenesis by BP3B in PDTX model. To measure the status of tumor angiogenesis in BP3B-treated tumors, immunofluorescence analysis of CD31 was performed. CD31 positive cells were visualized into red and nuclei were stained with DAPI (blue). The intensity of image was quantified with ImmunoRatio software. Scale bars are 50 μm. * p < 0.05