Upregulation of PIK3IP1 monitors the anti-cancer activity of PI3Kα inhibitors in gastric cancer cells.

Gastric cancer remains one of the most malignant cancers in the world. The target-based drugs approved by FDA for gastric cancer treatment include only three targets and benefit a small portion of gastric cancer patients. PIK3CA, a confirmed oncogene, mutates in 7-25% gastric cancer patients. PI3Kα inhibitor BYL719 has been approved for treating specific breast cancer. However, there is no comprehensive study about PI3Kα inhibitor in gastric cancer. In this study, we found pharmacological inhibition or knockdown of PI3Kα effectively inhibited the proliferation of partial gastric cancer cells. Then, we systematically explored the potential biomarkers for predicting or monitoring treatment response according to previous reports and found that basal expression of several receptor tyrosine kinases were related with the sensitivity of gastric cancer cells to BYL719. Next, RNA-seq technique was utilized and showed that BYL719 inhibited Myc targets V2 gene set in sensitive gastric cancer cells, and western blotting further verified that c-Myc was only inhibited in sensitive gastric cancer cells. More importantly, we firstly found BYL719 significantly elevated the expression of PIK3IP1 in sensitive gastric cancer cells, which was also observed in NCI-N87 cell derived xenograft mice models. Meanwhile, knockdown of PIK3IP1 partially rescued the cell growth inhibited by BYL719 in sensitive gastric cancer cells, suggesting the important role of PIK3IP1 in the antitumor activity of BYL719. In conclusion, our study provides biological evidence that PI3Kα is a promising target in specific gastric cancer and the elevation of PIK3IP1 could supply as a biomarker that monitoring treatment response.

[Effects of mTOR siRNA on mTOR/p70S6K signaling pathway in esophageal squamous cell carcinoma cells and the growth of transplanted tumor in nude mice].

OBJECTIVE: To investigate the effects of mTOR siRNA on mTOR-p70S6K signaling pathway in esophageal squamous cell carcinoma (ESCC) cells in vitro,and growth and apoptosis in transplanted tumor in nude mice. METHODS: mTOR siRNA was transfected into ESCC cell line EC9706 cells. The expressions of factors of the mTOR/p70S6K signaling pathway were detected by RT-PCR and Western blot. DNA contents and cell apoptosis were determined by flow cytometry, and cell proliferation was measured by CCK-8 assay. The effects of mTOR siRNA on the transplanted tumor growth were assessed in nude mice. RESULTS: The levels of mTOR and p-p70S6K were significantly decreased (P < 0.05) while the level of p70S6K was increased (P < 0.05) in the cells transfected with mTOR siRNA, compared with that in untransfected cells and cells transfected with control siRNA. After being interfered by mTOR siRNA, the number of apoptotic cells was increased, cell proliferation became slower and cell cycle was arrested in G(1) phase compared with that in control cells. Also, mTOR siRNA inhibited the growth of transplanted tumor in vivo. CONCLUSIONS: mTOR siRNA can effectively interfere in mTOR-p70S6K signaling pathway, induce cell apoptosis and inhibit cell proliferation and tumor growth, suggesting that mTOR-p70S6K signaling pathway plays an important role in the carcinogenesis and development of esophageal squamous cell carcinoma.

[Expression of Notch1 gene in esophageal squamous cell carcinoma cell line EC9706 and its effect on cell apoptosis].

BACKGROUND & OBJECTIVE: Notch1 signaling pathway is closely associated with carcinogenesis and plays a key role in cell growth, proliferation, differentiation, and apoptosis. This study was to investigate the expression of Notch1 gene in esophageal squamous cell carcinoma (ESCC) cell line EC9706 and its effects on cell apoptosis. METHODS: The expression of Notch1 gene in EC9706 cells was detected by immunocytochemistry. Notch1 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR). A eukaryotic expression vector pcDNA3.1-Notch1 (termed pcNICD) was constructed and transfected into EC9706 cells; pcDNA3.1 was also transfected as control. After transfection, the expression of Notch1 in EC9706 cells was detected by RT-PCR and Western blot, cell apoptosis was measured by flow cytometry (FCM). RESULTS: The expression of Notch1 gene was detected in EC9706 cells. The mRNA and protein expression of Notch1 gene in pcNICD-transfected EC9706 cells were significantly increased by about 3 folds of those in untreated and pcDNA3.1-transfected cells (P<0.01). However, there was no difference in Notch1 expression between untreated and pcDNA3.1-transfected EC9706 cells (P>0.05). The apoptosis rate was significantly higher in pcNICD-transfected EC9706 cells than in untreated and pcDNA3.1-transfected cells (P<0.01); there was no prominent difference between untreated and pcDNA3.1-transfected EC9706 cells (P>0.05). CONCLUSION: The activated Notch1 signaling pathway gives rise to the apoptosis of EC9706 cells, suggesting that Notch1 gene may be a new therapeutic target for ESCC.

[TAp63gamma-induced apoptosis mediated by apoptosis inducing factor in human esophageal squamous carcinoma EC9706 cells].

OBJECTIVE: To study the molecular mechanism of TAp63gamma-induced cell apoptosis. METHODS: Transcription and protein expression of apoptosis inducing factor and p63 were investigated by immunohistochemistry and RT-PCR in human esophageal squamous carcinoma cell line EC9706 respectively. Twenty-four hours after transfection with pcDNA3.1-TAp63gamma, the apoptosis and translocation of apoptosis inducing factor in EC9706 cells were studied by flow cytometry, laser confocal microscopy and mitochondrial/cytosol/nuclear extraction analysis respectively. Down-regulation of apoptosis inducing factor protein was achieved by RNAi and pretreatment with caspase inhibitor zVAD.fmk of EC9706 cells. RESULTS: Presence of protein expressions of apoptosis inducing factor and absence of TAp63gamma was observed in the cytoplasm of untransfected cells. RT-PCR verified the subtype of p63 in EC9706 cells was DeltaNp63. After 24 hours of transfection, both nuclear and cytoplasmic expression of apoptosis inducing factor protein were observed in cells transfected with TAp63gamma and p53 expression vectors, but not in cells transfected with control vector. Cell apoptosis rates were 1.37%, 13.64%, 4.52%, 4.03% and 1.91% in the pcDNA3.1 transfection group, pcDNA3.1-TAp63gamma transfection group, apoptosis inducing factor siRNA and pcDNA3.1-TAp63gamma transfection group, zVAD.fmk treatment group, and the group receiving apoptosis inducing factor siRNA, plus zVAD.fmk treatment and pcDNA3.1-TAp63gamma transfection, respectively. CONCLUSIONS: Apoptosis inducing factor of EC9706 cells is released from mitochondria into both the cytoplasm and nucleus during TAp63gamma induced apoptosis. Down-regulation of apoptosis inducing factor inhibits TAp63gamma-induced apoptosis. Overall, TAp63gamma-induced apoptosis is dependent on the expression of apoptosis inducing factor and caspase.

[Cloning and analysis of psaB cDNA of Dunaliella salina].

One pair of degenerate primer was designed according to conserved motifs of the psaB (A2 subunit of photosystem I) of Chlamydomonas reinhardtii, Chlamydomonas moewusii, Chlorella vulgaris and Mesostigma viride, and a total RNA of Dunaliella salina (D. salina) was extracted with TRIzol reagent. A cDNA fragment, about 1.8kb in length, from green algal D. salina was obtained through RT-PCR method. The resulting PCR product was cloned into T-vector and screened to determine its sequence. Homologous analysis of the deduced amino acid sequence was performed by BLAST and subsequeqtly compared with GenBank data. The obtained cDNA sequence was 1815 bp long, which encodes 605 amino acids (GenBank accession number: AY820754). The sequence shared high homologue with the following psaB: Chlamydomonas reinhardtii 92%, Chlamydomonas moewusii 91%, Chlorella vulgaris 86%, Mesostigma viride 85%, Physcomitrella patens subsp. Patens 85% and Nephroselmis olivacea 84%. It can be concluded that the cloned sequence is psaB cDNA fragment from D. salina.

[Construction of randomly matrix attachment regions library from the green alga: Dunaliella salina].

Matrix attachment region (MAR) is DNA fragment that can bind to the nuclear matrix. In order to isolate the MAR fragment from the halotolerant green alga Dunaliella salina, we created a library of randomly obtained MAR from D. salina. Firstly the intact nuclei were released using 0.5% Triton X-100, then purification was carried out by discontinuous centrifugation using 30% and 70% Percoll gradients. Histones of nuclear matrices were removed using 25mmol/L lithium dioodosalicylate, the DNAs not closely associated with the matrices were removed using restriction enzymes. The remained matrices DNAs were digested by proteinase K, extracted with phenol/chloroform and precipitated with ethanol, and then cut with four kinds of restriction enzymes, the resulting DNAs were subsequently ligated to pUC18-vector and transferred to E. coli JM109 strains, DNA sequencing showed that the DNA fragments had the features of MAR DNA fragments.

[Cloning and functional analyses of promoters of two carbonic anhydrase genes from Dunaliella salina].

The cloning vectors pMD-DCA1 and pMD-CA containing the promoters of duplicated carbonic anhydrase 1 (DCA1) and carbonic anhydrase (CA) genes, respectively, from Dunaliella salina, and expression vector pDM307 containing bar-NOS polyA fragment were digested with EcoR I. The bar-NOS polyA fragment was fused, respectively, to the fragments of the vectors pMD-DCA1 and pMD-CA to form transgenic D. salina expression vectors pMDDC-B and pMDC-B. The micro-shots were prepared by coating two constructs (pMDDC-B and pMDC-B) with gold particle. Each sample was bombarded once, twice, and thrice, respectively, with micro-projectile gun at a rupture pressure of 690 kPa in helium gas. The screening culture of the bombarded alga cells was performed in PKS liquid and solid medium containing 3 mg/L phosphinothricin (PPT) to develop the transformed cells of D. salina. Analyses of the transformed cells were carried out through PCR, Southern blotting, and Northern blotting. The results of screening culture showed that the expression of the external bar gene of vectors pMDDC-B and pMDC-B was stable and transient, respectively, in the transformed D. salina cells. In the meantime, the transformed efficiency of particle bombardment twice was higher than that of once or thrice particle bombardment at a rupture pressure of 690 kPa in helium gas. PCR and Southern blotting analyses indicated that the external bar gene was integrated into the genome of the cells. Northern analysis indicated that expression efficiency of the bar gene driven by DCA1 promoter was regulated by the gradient concentration of sodium chloride, and the positive blotting signal intensity of the bar mRNA was highest in the medium containing 2 mol/L of sodium chloride. The findings of the present study suggest that promoter of the DCA1 gene may be an inducible promoter following a hyperosmotic shock with high activity and safety in the research of transgenic D. salina. The tandem GT sequences of the promoter region of DCA1 and CA genes may be related to the molecular mechanisms of the extreme halo-toleration of the unicellular green alga, D. salina.

[Expression of mouse canstatin and its N-domain in E. coli BL21].

The mouse canstatin and its N-domain cDNA were amplified from total RNA of mouse liver by RT- PCR and cloned into vector pMD18-T for sequencing. Prokaryotic expression vectors pET/Can and pET/Can-N were constructed and expressed in E.coli BL21(DE3) with induction of IPTG.. Mouse canstatin cDNA is 684bp in length encoding 227 amino acids. The sequences of both cDNA and amino acids share high homology with human canstatin, with cDNA identity at 89% and amino acids identity at 96% to human canstatin. N-domain of mouse canstatin is the same amino acid sequence as that of human canstatin. In the present study, prokaryotic expression vector pET/Can and pET/Can-N were expressed in E.coli BL21 with amount of 35% and 18% of the total bacterial proteins after being induced by IPTG for 4h. The expressed products existed mainly as inclusion bodies. This work has laid down the basis for further study of its angiogenic activity and potential application for tumor dormancy therapy.