Downregulated NM23-H1 expression is associated with intracranial invasion of nasopharyngeal carcinoma

Abstract

Because the focus of nasopharyngeal carcinoma (NPC) is very close to intracranial organs, it often makes incursions into cranial cavity. Identification of intracranial invasion-associated indicators will provide potential therapeutic targets for NPC patients with intracranial invasion. In this regard, Human Xpro HC-plus cancer-related gene chip was utilised to screen intracranial invasion-associated genes for NPC from the biopsied primary focus tissue samples. In all, 8 upregulated and 23 downregulated genes were obtained. VEGF165 and MMP-9, the two upregulated genes, and NM23-H1, the downregulated one, were further confirmed by immunohistochemistry, quantitative real-time PCR and western blot. Invasion-associated cellular and nude mouse models were subsequently employed to study the biological properties of NM23-H1. NM23-H1 expression was significantly lower in 5-8F cells compared with that in 6-10B cells. Moreover, patch-clamp and transwell chamber were adopted to investigate the invading potential-associated biological dynamic mechanisms in the two cell lines, and Ca(2+) current and motility were significantly elevated in 5-8F cells compared with that in 6-10B cells. Berberine, an inhibitor of Ca(2+) current, could substantially increase the expression of NM23-H1 and decrease 5-8F cell motility. The specificity of berberine on NM23-H1 and cell motility was confirmed by RNAi assay.

Figure 1

Analysis of the differentially expressed genes associated with intracranial invasion in primary NPC. Note: C5-NPC group with intracranial invasion; C1-NPC group without invasion. (A) No invading NPC tissues hybridised atlas of Cy3 and cRNA Xpro HC-III plus labelled; (B) Intracranial invading NPC tissues hybridised atlas of Cy3 and cRNA Xpro HC-III plus labelled; the gene-expressing profiling was done three times for each group, and six chips have been performed for the 20 patients. (C and D) Data normalised scatter plot from the NPC tissue samples with intracranial invading and those with no invasion other than the primary focus. Systemic difference caused by the process of lab was decreased by normalising the compared data. (E) Analysing of hybridisation scatter plot of differentially expressive genes in the NPC tissue samples with intracranial invading tendency and those without invasion outside the primary focus. In the panels C, D and E showing the natural logarithm of data from two compared groups as X-axis and Y-axis, difference of two compared groups was judged directly by distribution tendency. The genes distributed along 45° diagonal have been equally expressed, and for those, the vertically further to diagonal, the greater difference was shown in their differential expression.

Figure 2

Validation of NM23-H1, VEGF165 and MMP-9 expression in the tissue samples of primary focus of NPC. (A) Validation with IHC (H&E, × 10). A1, A3, A5: NPC tissues with no invasion; A2, A4, A6: NPC tissues with intracranial invading signs. (B) Validation by the quantitative real-time PCR. Glyceraldehyde 3-phosphate dehydrogenase served as control. Relative mRNA levels of NM23-H1, VEGF165 or MMP-9 mRNA/GAPDH are expressed as the relative abundance. C1: NPC tissues with no invasion, C5: NPC tissues with intracranial invading signs. (C) Validation with western blot. Tissues lysates from two groups were subjected to western immunoblotting with anti-NM23-H1, VEGF165 or MMP-9 antibody, and blot was reprobed with anti-GAPDH to verify equal loading.

Figure 3

The inhibitory effect of berberine on the NPC invasion in vitro and in vivo. Cytotoxic effects of berberine on 5-8F (A), 6-10B (B) or siNM23-H1 transfected 5-8F cells (C). The cytotoxicity of berberine was evaluated using an XTT cell proliferation assay. 5 × 10⁴ cells were cultured for 24, 48, 72 and 96 h in 96-well microplates (100 μl per well) with various concentration of berberine as indicated. Cell viability values (%) are expressed as means±s.e. from four separate measurements, t-test. (D) Inhibitory effect of berberine on cell motility in 5-8F, 6-10B and siNM23-H1 transfected 5-8F cells (cultured with 2.8 μg ml⁻¹ berberine for 72 h). The values (% of cells) are expressed as means±s.e. from four separate measurements, t-test, ^*P<0.01, 5-8F, berberine-treated siNM23-H1-transfected 5-8F cells vs other cells. (E) Berberine increased NM23-H1protein expression in NPC cells, and siNM23-H1 is effective in knocking down the expression of endogenesis NM23-H1. (F, G) Berberine inhibited I_CRAC in NPC cells. Igor Pro 4.0 demo was used to observe and record the effect of prolonging time on I_CRAC. (H) Left panel: Berberine increased NM23-H1 expression in vivo. NM23-H1 mRNA in the implanting tumour tissues of nude mice was shown as the relative abundance and GAPDH as control. Right panel: Berberine increased NM23-H1 protein expression in vivo. Berberine (200 mg kg⁻¹ day⁻¹) was injected into different groups of nude mice with implanting tumour induced by injection of various lines of NPC cells. NM23-H1 expression level was significantly higher in the tumour tissues from animals induced with 5-8F than those with 6-10B cells.