Effect Of Anticancer Peptide ANP Expressed By Recombinant Adenovirus On Tongue Squamous Cell Carcinoma

Anticancer peptide is a new anticancer polypeptide drugs with small molecular,which can kill cancer cells by destroying the structure of tumor cell membrane,inhibiting the proliferation and migration of cancer cells and the formation of tumor blood vessels.It has become a hot spot of research on new anti-tumor drugs.Studies have shown that ANP,Atrial peptide hormone,can strongly inhibit the synthesis of DNA in cancer cells,thereby inhibiting cell growth.ANP has no harm to normal human cells and also is able to alleviate the toxicity of anti-cancer drugs on the kidney and liver.ANP expressed in cancer cells can not only solve the problem of short half-life peptides,but also avoid the side effects on other cells.Using the gene therapy,mammalian cells,infected with recombinant adenovirus?rAd?,secreting the anti-cancer peptide ANP.ANP and cancer cells can achieve“contacted closely”,overcoming the shortcoming that anti-cancer drugs have trouble reaching the target.At the same time,the anti-cancer peptide ANP can be continuously expressed in cancer tissue cells to kill cancer cells efficiently.In this study,specific primers were designed according to the cDNA sequence of ANP.By extracting intracellular mRNA,cDNA was obtained by reverse transcription and PCR.The overlapping PCR primers carrying Ig?signal peptide were designed to obtain the Ig?signal peptide gene by overlapping PCR.The ANP and IgANP gene were then obtain by overlapping PCR again.After the gene was connected to the T vector and identified by sequencing correctly,the ANP gene and IgANP gene were respectively connected to the pAd-Track-CMV shuttle vector.Finally,the linearized pAd-Track-ANP and adenovirus skeleton vector,pAd-easy,were transformed to BJ5183together for homologous recombination to obtain the pAd-ANP and pAd-IgANP rAd vectors.The rAd vectors were then transfected into 293A cells to get adenovirus.The virus genome containing ANP was identified by PCR.After rAd infected SCC9 cells,RT-PCR was carried out to identify the expression of ANP gene.F1 generation rAd was continuously amplified to F5 generation.The titer of rAd of F5 generation were 10⁸PFU/ml and the VP of Ad-EGFP,Ad-ANP?Ad-IgANP were 8.5×10¹¹,5.4×10¹¹and6.1×10¹¹,respectively.These rAd can be used in the following experiments.Flow cytometry was used to detect infection efficiency and the optimal MOI of rAd in tongue squamous cell carcinoma was 100.Immunofluorescence showed that ANP was expressed in tongue squamous carcinoma cells,and MTT assay showed that the relative survival rate of SCC9 cells infected with Ad-ANP was about 79.9%,82.0%and 82.9%at 48h,72h and 96h,and that of SCC25 cells was 97.1%,94.0%and 79.5%respectively.The relative survival rate of SCC9 cells infected with Ad-IgANP was about89.9%,93.8%and 90.2%at 48h,72h and 96h and SCC25,98.9%,91.9%and 85.9%respectively.The colony formation experiment showed that the inhibition rate of Ad-ANP on SCC9 cells was 36.9%and that of SCC25 cells was 62.9%.The inhibition rate of Ad-IgANP on SCC9 cell cloning was 22.3%and on SCC25 was 34.6%.Scratch test showed that the Ad-ANP reduced by 67.8%at 72h.There was decreased by 47.9%and 37.6%in SCC25 cells at at 48h and 72h respectively.The Ad-IgANP had decreased by 40.9%and 58.7%at 24h and 72h respectively.Migration rate of SCC25cells decreased by 46.9%and 34.2%at 48h and 72h respectively.Flow cytometry instrument detected the cell cycle and apoptosis,which showed that the Go/G1 phase,S phase and G2/M phase accounted for 59.7%,19.6%and 17.7%while Ad-EGFP processing SCC9 cells and 59.1%,25.0%and 15.8%while Ad-EGFP processing SCC25cells.Go/G1 phase,S phase and G2/M phase accounted for 67.0%,11.9%and 17.7%while Ad-ANP processing SCC9 cells and 62.1%,23.2%and 14.7%while Ad-ANP processing SCC25 cells.Go/G1 phase,S phase and G2/M phase accounted for 57.6%,18.1%and 19.3%while Ad-IgANP processing SCC9 cells and 60.9%,29.8%and 9.3%while Ad-IgANP processing SCC25 cells.ANP expressed by Ad-ANP increased the apoptosis of SCC9 and SCC25 cells by 65.9%and 57.9%respectively.ANP expressed by Ad-IgANP increased the apoptosis of SCC9 and SCC25 cells by 61.6%and 8.7%respectively.Western blot analysis tested the intracellular protein expression after rAd infection,which showed that Ad-ANP and Ad-IgANP up-regulated PARP,caspase8 and BAX protein of SCC9 cell,while the expression of caspase3 did not change significantly.It has been confirmed that the Ad-ANP and Ad-IgANP expressing ANP intracellularly may increase the apoptosis of SCC9 and SCC25 of tongue phosphorous squamous carcinoma cells through the caspase8 signal pathway.In this stduy,we found anticancer peptide ANP expressed by recombinant adenovirus,have obvious influence on the proliferation and apoptosis of tongue cancer cells SCC9 and SCC25.The possible mechanism of tongue cancer cell growth inhibiting by ANP maybe caused by the apoptosis of the mitochondrial pathways.Thereby,gene therapy is a promising strategy to express anti-cancer peptides ANP in tongue cancer therapy by adenovirus recombination in the future...