Effects of Zeng Sheng Ping/ACAPHA on malignant brain tumor growth and Notch signaling

Abstract

Background/aim: Zeng Sheng Ping (ZSP) is a traditional herbal remedy used to prevent progression and growth of neoplastic lesions. It has been shown to inhibit Notch2 expression in a murine lung cancer model, leading us to investigate its therapeutic potential in Notch-dependent brain tumors.

Materials and methods: 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), apoptosis, and quantitative real-time polymerase chain reaction (RT-PCR) analyses were performed in glioma and medulloblastoma cell lines, and morphological analyses in DAOY flank xenografts.

Results: ZSP inhibited brain tumor growth in vitro, in part, by apoptotic induction. Down-regulation of the Notch2 receptor, the pathway target Hairy/Enhancer of Split homolog 1 (Hes1), and of the stem cell markers Nestin and CD133 was also observed. Reductions in tumor mass and increases in the necrotic fraction of DAOY xenografts, in mice treated with oral ZSP were also observed, but these were not significant.

Conclusion: ZSP can block brain tumor growth and the expression of Notch pathway members and stem cell markers in vitro.

Figure 1

Zeng Sheng Ping (ZSP) inhibits brain tumor cell growth. ZSP treatment reduced tumor cell mass as measured by MTS assay in the medulloblastoma cell line DAOY (Figure 1A), as well as the glioblastoma cell lines U87 (B), HSR-GBM1 (C), JHH-GBM10 (D) and JHH-GBM14 (E). ***p≤ 0.0001 relative to mock-treated control.

Figure 2

ZSP induces apoptotic cell death in a subset of brain tumor cell lines. After adherent medulloblastoma and glioblastoma cell lines DAOY (A and B) and U87 (C) were subjected to ZSP treatment, a dose-dependent increase in the percentage of apoptosis was observed. In the glioblastoma neurosphere lines HSR-GBM1 (D), JHH-GBM10 (E), and JHH-GBM14 (F), the percentage of dead cells increased in a dose-dependent fashion. *p≤0.05, **p≤0.001 and ***p≤0.0001 relative to mock-treated control.

Figure 3

ZSP inhibits Notch signaling and cancer stem-like marker expression in DAOY and U87 cells. Transcript levels of the Notch1 receptor remain unchanged (A and B) while those of Notch2 receptor were down-regulated following ZSP treatment in DAOY (C) and U87 (D) cells. Hes1, a Notch downstream target, also showed dose-dependent reductions (E and F). In addition, transcript levels of stem-like cancer markers, Nestin and CD133, also demonstrated statistical significant reductions at higher concentrations (G–J). HPRT: hypo-xanthine phosphoribosyltransferase. *p≤0.05, **p≤0.01 and ***p≤0.0001 relative to the mock-treated control.

Figure 4

ZSP and medulloblastoma xenograft growth. In vivo oral ZSP treatment in nude mice with flank DAOY xenografts led to a reduction in tumor growth in the early treatment model (A) but the results were not statistically significant. No growth reduction was seen in the late treatment model (B). Microscopic examination revealed necrotic areas (*) in the xenografts (C, left six panels, original magnification ×40), which increased in size with treatment, but again these changes were not significant (D and E). Mitotic figures were also common (C, right two panels, arrows. original magnification ×400), but blinded counts showed only a non-significant reduction in this marker of proliferation (F and G). HPF: High power field; V: vehicle control