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Published on: October 30, 2013
Expression of Momordica charantia MAP30 and its antitumor effect on bladder cancer cells
Hao Hlin1, Zhang Zhi-Guo, Han Cong-Hui
1Department of Urology, Xuzhou Central Hospital, Xuzhou, Jiangsu Province, China - zpying58@126.com.
Background:
Momordica charantia (MC) is an edible medicinal plant that is known for its diversified biological functions. Momordica Antiviral Protein 30kD (MAP30) is a type I single chain ribosome-inactivating protein (RIP) isolated from the mature fruit and seeds of MC. Since MAP30 content in MC is limited, the study aim was to generate the recombinant MAP30 protein using prokaryotic expression system and determine its apoptotic/growth inhibitory effects on bladder cancer 5637 cells.
Methods:
MAP30 gene was amplified by PCR from MC genomic DNA and identified by sequencing. The target gene was inserted into pET-28a (+) vector and transformed into E. coli BL21 (DE3) cells. Positive clones were selected by PCR. Recombinant protein was efficiently expressed under induction with 1.0 mM Isopropylthio-β-D-galactoside (IPTG) at 30° C for 4 hours. Cytotoxicity studies were performed using MTT assay by treating 5637 bladder cancer cells with 100 µg/mL, 200 µg/mL, and 400 µg/mL concentrations of MAP30 for 24 hours and 48 hours, respectively. Flow cytometry was used to measure the apoptosis of MAP30-treatedcells in time course experiments.
Results:
Full-length MAP30 gene was successfully expressed in Escherichia coli (E. coli) BL21 strain and MAP30 recombinant protein inhibited the growth of bladder cancer 5637 cells at 200 µg/mL and 400 µg/mL concentrations by inducing apoptosis of target cells in a dose- and time-dependent manner.
Conclusion:
It was, therefore, concluded that the MAP30 recombinant protein displayed potent antitumor activity in vitro.
Insights
Recombinant Momordica Antiviral Protein 30kD (MAP30) effectively inhibited bladder cancer cell growth. This study demonstrates MAP30
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- Momordica charantia (MC) is a medicinal plant with diverse biological functions.
- Momordica Antiviral Protein 30kD (MAP30) is a ribosome-inactivating protein (RIP) from MC.
- Limited natural MAP30 necessitates recombinant protein production.
Purpose of the Study:
- To produce recombinant MAP30 using a prokaryotic system.
- To evaluate the apoptotic and growth inhibitory effects of recombinant MAP30 on bladder cancer 5637 cells.
Main Methods:
- MAP30 gene amplification, sequencing, and cloning into pET-28a (+) vector.
- Expression of recombinant MAP30 in E. coli BL21 (DE3) under IPTG induction.
- MTT assay for cytotoxicity and flow cytometry for apoptosis analysis.
Main Results:
- Successful expression of full-length MAP30 in E. coli.
- MAP30 inhibited bladder cancer 5637 cell growth at 200 and 400 µg/mL.
- Apoptosis induction was dose- and time-dependent.
Conclusions:
- Recombinant MAP30 exhibits potent in vitro antitumor activity.
- MAP30 is a promising candidate for bladder cancer therapy.
