Pinellia pedatisecta agglutinin interacts with the methylosome and induces cancer cell death

Abstract

Pinellia pedatisecta agglutinin (PPA) is a specific mannose-binding plant lectin accumulated in the tuber of P. pedatisecta. In the work presented, the cytotoxicity of PPA to cancer cells was investigated through exogenous expression. A PPA gene was transduced into normal and cancer cell lines through plasmid vectors, and the effect of PPA expression was examined. Results showed that PPA translocated into the nucleus, colocalized with DNA and induced cell death. A mannose-binding motif and a V(103)-W(130) region directed the nuclear translocation of PPA. Coprecipitation, mass spectrometry and western blotting analysis further indentified that PPA was associated with the methylosome, which contains methylosome protein 50 and protein arginine methyltransferase 5 (PRMT5). Knockdown of PRMT5 significantly inhibited the PPA-induced cell death, suggesting that PPA used the methylosome as a target. Furthermore, Ad.surp-PPA, an adenovirus vector in which the PPA gene was controlled by a survivin promoter (surp), selectively inhibited the proliferation of cancer cell lines. Taken together, the expression of PPA gene elicited significant cytotoxicity to cancer cells through targeting the methylosome and might be developed into a novel agent in cancer gene therapy.

Figure 1

The amino acid sequence and exogenous expression of PPA in human cells. (a) The amino acid sequence of PPA. Underlined amino acid sequences show three MBMs. (b) The exogenous expression of his-PPA in a normal lung cell line WI38 and lung cancer cell lines A549, H460 and H1299. Cells were transfected with pcDNA3.1/His or pcDNA3.1/His-PPA. Cell lysates were analyzed by western blot with antibodies against 6His or Actin. (c) The exogenous expression of his-PPA in liver cancer cell lines PLC or Hep3B. Cells were transfected with pcDNA3.1/His or pcDNA3.1/His-PPA. Cell lysates were analyzed by western blot with antibodies against 6His or Actin. (d) The exogenous expression of FLAG-PPA in H1299 cells. Cells were transfected with pcDNA3/FLAG or pcDNA3/FLAG-PPA. Cell lysates were analyzed by western blot with antibodies against FLAG or Actin.

Figure 2

PPA translocated into the nucleus and induced cell death. Cells were transfected with pEGFP-C1 or pEGFP-PPA-C1 followed by fluorescent microscope observation after 24 h (early stage) or 48 h (late stage). Cells transfected with pEGFP-PPA-C1 were further stained with propidium iodide after 48 h. Bars show 100 μm.

Figure 3

Regions in directing the nuclear translocation of PPA. (a) Cells were transfected with pEGFP-MBM-C1 and pEGFP-C1 followed by fluorescent microscope observation after 48 h. (b) H1299 cells transfected with pEGFP-MBM-C1 were further stained with Hoechst33342 after 48 h followed by fluorescent microscope observation. (c) H1299 cells were transfected with pEGFP-C1 or pEGFP-(V¹⁰³-W¹³⁰)-C1 followed by fluorescent microscope observation after 48 h. Bars show 100 μm.

Figure 4

PPA-induced PARP level. H1299 and H460 cells were transfected with pcDNA3.1/His-PPA or pcDNA3.1/His-EGFP. After 48 h, cells lysates were analyzed by western blot with antibodies against caspase3, caspase8, PARP and Actin.

Figure 5

PPA-induced cell death through targeting the methylosome. (a) H1299 cells were transfected with pcDNA3/FLAG-PPA or pcDNA3/FLAG. Cell lysates were immunoprecipitated with a FLAG antibody conjugated gel. Precipitation with a normal mouse IgG plus a protein G conjugated agarose served as a control. The precipitated complexes were examined by western blotting analysis with antibodies against MEP50 and PRMT5. Actin, MEP50 and PRMT5 in whole-cell lysates was monitored for expression levels. (b) H1299 cells were transfected with a control siRNA or PRMT5 siRNA followed by a second transfection with pEGFP-PPA-C1 after 48 h. Bars show 100 μm. (c) H1299 cells were transfected with a control siRNA or PRMT5 siRNA. After 48 h, the whole-cell lysates were examined by western blot with antibodies against PRMT5 and actin.

Figure 6

Ad.surp-PPA elicited a selective toxicity to cancer cells. Normal lung cell line WI38, lung-cancer cell line H1299 and liver cancer cell lines PLC and Huh7 were treated with Ad or Ad.surp-PPA at multiplicity of infections indicated. After 48 h, cell viability was analyzed by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Values are shown as mean±s.d.