Delivery of miR-3529-3p using MnO2 -SiO2 -APTES nanoparticles combined with phototherapy suppresses lung adenocarcinoma progression by targeting HIGD1A.

BACKGROUND: The present study aimed to investigate the function of miR-3529-3p in lung adenocarcinoma and MnO2 -SiO2 -APTES (MSA) as a promising multifunctional delivery agent for lung adenocarcinoma therapy. METHODS: Expression levels of miR-3529-3p were evaluated in lung carcinoma cells and tissues by qRT-PCR. The effects of miR-3529-3p on apoptosis, proliferation, metastasis and neovascularization were assessed by CCK-8, FACS, transwell and wound healing assays, tube formation and xenografts experiments. Luciferase reporter assays, western blot, qRT-PCR and mitochondrial complex assay were used to determine the targeting relationship between miR-3529-3p and hypoxia-inducible gene domain family member 1A (HIGD1A). MSA was fabricated using MnO2 nanoflowers, and its heating curves, temperature curves, IC50, and delivery efficiency were examined. The hypoxia and reactive oxygen species (ROS) production was investigated by nitro reductase probing, DCFH-DA staining and FACS. RESULTS: MiR-3529-3p expression was reduced in lung carcinoma tissues and cells. Transfection of miR-3529-3p could promote apoptosis and suppress cell proliferation, migration and angiogenesis. As a target of miR-3529-3p, HIGD1A expression was downregulated, through which miR-3529-3p could disrupt the activities of complexes III and IV of the respiratory chain. The multifunctional nanoparticle MSA could not only efficiently deliver miR-3529-3p into cells, but also enhance the antitumor function of miR-3529-3p. The underlying mechanism may be that MSA alleviates hypoxia and has synergistic effects in cellular ROS promotion with miR-3529-3p. CONCLUSIONS: Our results establish the antioncogenic role of miR-3529-3p, and demonstrate that miR-3529-3p delivered by MSA has enhanced tumor suppressive effects, probably through elevating ROS production and thermogenesis.

Oncogenic TRIB2 interacts with and regulates PKM2 to promote aerobic glycolysis and lung cancer cell procession.

PKM2 is an important regulator of the aerobic glycolysis that plays a vital role in cancer cell metabolic reprogramming. In general, Trib2 is considered as a "pseudokinase", contributing to different kinds of cancer. However, the detailed roles of TRIB2 in regulating cancer metabolism by PKM2 remain unclear. This study demonstrated that TRIB2, not a "pseudokinase", has the kinase activity to directly phosphorylate PKM2 at serine 37 in cancer cells. The elevated pSer37-PKM2 would subsequently promote the PKM2 dimers to enter into nucleus and increase the expression of LDHA, GLUT1, and PTBP1. The aerobic glycolysis is then elevated to promote cancer cell proliferation and migration in TRIB2- or PKM2-overexpressed cultures. The glucose uptake and lactate production increased, but the ATP content decreased in TRIB2- or PKM2-treated cultures. Experiments of TRIB2-/- mice further supported that TRIB2 could regulate aerobic glycolysis by PKM2. Thus, these results reveal the new kinase activity of TRIB2 and its mechanism in cancer metabolism may be related to regulating PKM2 to promote lung cancer cell proliferation in vitro and in vivo, suggesting promising therapeutic targets for cancer therapy by controlling cancer metabolism.

miR-4293 upregulates lncRNA WFDC21P by suppressing mRNA-decapping enzyme 2 to promote lung carcinoma proliferation.

Non-coding RNAs (ncRNAs) involve in diverse biological processes by post-transcriptional regulation of gene expression. Emerging evidence shows that miRNA-4293 plays a significant role in the development of non-small cell lung cancer. However, the oncogenic functions of miR-4293 have not been studied. Our results demonstrated that miR-4293 expression is markedly enhanced in lung carcinoma tissue and cells. Moreover, miR-4293 promotes tumor cell proliferation and metastasis but suppresses apoptosis. Mechanistic investigations identified mRNA-decapping enzyme 2 (DCP2) as a target of miR-4293 and its expression is suppressed by miR-4293. DCP2 can directly or indirectly bind to WFDC21P and downregulates its expression. Consequently, miR-4293 can further promote WFDC21P expression by regulating DCP2. With a positive correlation to miR-4293 expression, WFDC21P also plays an oncogenic role in lung carcinoma. Furthermore, knockdown of WFDC21P results in functional attenuation of miR-4293 on tumor promotion. In vivo xenograft growth is also promoted by both miR-4293 and WFDC21P. Overall, our results establish oncogenic roles for both miR-4293 and WFDC21P and demonstrate that interactions between miRNAs and lncRNAs through DCP2 are important in the regulation of carcinoma pathogenesis. These results provided a valuable theoretical basis for the discovery of lung carcinoma therapeutic targets and diagnostic markers based on miR-4293 and WFDC21P.

Celastrol suppresses the proliferation of lung adenocarcinoma cells by regulating microRNA-24 and microRNA-181b.

Cumulative evidence has indicated that celastrol may suppress cancer growth; however, the underlying mechanism requires further investigation. In the present study, A549 cells were treated with various concentrations of celastrol. Lung cancer cell proliferation was evaluated using an MTT assay and observed under a microscope. Cell apoptosis was detected by Annexin V fluorescein isothiocyanate/propidium iodide double-labeled flow cytometry. The results demonstrated that celastrol suppressed proliferation and induced apoptosis in a dose-independent manner. Celastrol may also decrease the phosphorylation levels of signal transducer and activator of transcription 3 (STAT3) and the B cell lymphoma-2 (Bcl-2)/Bcl-2 associated C protein (Bax) ratio. As microRNA (miR-24 and miR-181b) were predicated to target STAT3, STAT3 activation was inhibited in miR-24-or miR-181b-treated A549 cells compared with the control treatment. The ratio of Bcl-2/Bax was further reduced in miR-24 or miR-181b-treated A549 cells. The results were further confirmed by detecting in another lung adenocarcinoma cell line, LTEP-a-2. In summary, the results of the present study demonstrated that celastrol treatment suppressed the proliferation and induced apoptosis by regulating the expression levels of miR-24 and miR-181b.

MiR-320a effectively suppresses lung adenocarcinoma cell proliferation and metastasis by regulating STAT3 signals.

MicroRNAs play important roles in tumorigenesis of various types of cancers. MiR-320a can inhibits cell proliferation of some cancers, but the biologic roles of miR-320a in lung cancer need to be further studied. Here, we investigated the roles of miR-320a in suppressing the proliferation of lung adenocarcinoma cells. MiR-320a treatment was found to effectively suppress LTEP-a-2 and A549 cell proliferation, and induce more apoptotic cells with irradiation treatment compared with control treatment. Our results also showed that miR-320a, as a novel miRNA, directly regulated signal transducer and activator of transcription 3 (STAT3) and its signals, such as Bcl-2, Bax, and Caspase 3. The siRNA-inhibited STAT3 levels further proved its roles in regulating STAT3 signals. Moreover, miR-320a treatment effectively suppressed cancer cell growth in mice xenografts compared with controls, and significantly inhibited cell migration in vitro and in vivo. Our findings collectively demonstrated that miR-320a, by directly regulating STAT3 signals, not only suppressed cell proliferation and metastasis, but also enhanced irradiation-induced apoptosis of adenocarcinomia cells.

The Paraoxonase Gene Cluster Protects Against Abdominal Aortic Aneurysm Formation.

OBJECTIVE: Abdominal aortic aneurysm (AAA) is a life-threatening vascular pathology, the pathogenesis of which is closely related to oxidative stress. However, an effective pharmaceutical treatment is lacking because the exact cause of AAA remains unknown. Here, we aimed at delineating the role of the paraoxonases (PONs) gene cluster (PC), which prevents atherosclerosis through the detoxification of oxidized substrates, in AAA formation. APPROACH AND RESULTS: PC transgenic (Tg) mice were crossed to an Apoe-/- background, and an angiotensin II-induced AAA mouse model was used to analyze the effect of the PC on AAA formation. Four weeks after angiotensin II infusion, PC-Tg Apoe-/- mice had a lower AAA incidence, smaller maximal abdominal aortic external diameter, and less medial elastin degradation than Apoe-/- mice. Importantly, PC-Tg Apoe-/- mice exhibited lower aortic reactive oxidative species production and oxidative stress than did the Apoe-/- control mice. As a consequence, the PC transgene alleviated angiotensin II-induced arterial inflammation and suppressed arterial extracellular matrix degradation. Specifically, on angiotensin II stimulation, PC-Tg vascular smooth muscle cells exhibited lower levels of reactive oxidative species production and a decrease in the activities and expression levels of matrix metalloproteinase-2 and matrix metalloproteinase-9. Moreover, PC-Tg serum also enhanced vascular smooth muscle cell oxidative stress resistance and further decreased the expression levels of matrix metalloproteinase-2 and matrix metalloproteinase-9, indicating that circulatory and vascular smooth muscle cell PC members suppress oxidative stress in a synergistic manner. CONCLUSIONS: Our findings reveal, for the first time, a protective role of the PC in AAA formation and suggest PONs as promising targets for AAA prevention.

Smad3-related miRNAs regulated oncogenic TRIB2 promoter activity to effectively suppress lung adenocarcinoma growth.

MicroRNAs (miRNAs) and Smad3, as key transcription factors in transforming growth factor-ß1 (TGF-ß1) signaling, help regulate various physiological and pathological processes. We investigated the roles of Smad3-regulated miRNAs with respect to lung adenocarcinoma cell apoptosis, proliferation, and metastasis. We observed that Smad3 and phospho-SMAD3 (p-Smad3) were decreased in miR-206- (or miR-140)-treated cells and there might be a feedback loop between miR-206 (or miR-140) and TGF-ß1 expression. Smad3-related miRNAs affected tribbles homolog 2 (TRIB2) expression by regulating trib2 promoter activity through the CAGACA box. MiR-206 and miR-140 inhibited lung adenocarcinoma cell proliferation in vitro and in vivo by suppressing p-Smad3/Smad3 and TRIB2. Moreover, lung adenocarcinoma data supported a suppressive role for miR-206/miR-140 and an oncogenic role for TRIB2-patients with higher TRIB2 levels had poorer survival. In summary, miR-206 and miR-140, as tumor suppressors, induced lung adenocarcinoma cell death and inhibited cell proliferation by modifying oncogenic TRIB2 promoter activity through p-Smad3. MiR-206 and miR-140 also suppressed lung adenocarcinoma cell metastasis in vitro and in vivo by regulating EMT-related factors.

Age-Associated Sirtuin 1 Reduction in Vascular Smooth Muscle Links Vascular Senescence and Inflammation to Abdominal Aortic Aneurysm.

RATIONALE: Uncontrolled growth of abdominal aortic aneurysms (AAAs) is a life-threatening vascular disease without an effective pharmaceutical treatment. AAA incidence dramatically increases with advancing age in men. However, the molecular mechanisms by which aging predisposes individuals to AAAs remain unknown. OBJECTIVE: In this study, we investigated the role of SIRT1 (Sirtuin 1), a class III histone deacetylase, in AAA formation and the underlying mechanisms linking vascular senescence and inflammation. METHODS AND RESULTS: The expression and activity of SIRT1 were significantly decreased in human AAA samples. SIRT1 in vascular smooth muscle cells was remarkably downregulated in the suprarenal aortas of aged mice, in which AAAs induced by angiotensin II infusion were significantly elevated. Moreover, vascular smooth muscle cell-specific knockout of SIRT1 accelerated angiotensin II-induced formation and rupture of AAAs and AAA-related pathological changes, whereas vascular smooth muscle cell-specific overexpression of SIRT1 suppressed angiotensin II-induced AAA formation and progression in Apoe-/- mice. Furthermore, the inhibitory effect of SIRT1 on AAA formation was also proved in a calcium chloride (CaCl2)-induced AAA model. Mechanistically, the reduction of SIRT1 was shown to increase vascular cell senescence and upregulate p21 expression, as well as enhance vascular inflammation. Notably, inhibition of p21-dependent vascular cell senescence by SIRT1 blocked angiotensin II-induced nuclear factor-κB binding on the promoter of monocyte chemoattractant protein-1 and inhibited its expression. CONCLUSIONS: These findings provide evidence that SIRT1 reduction links vascular senescence and inflammation to AAAs and that SIRT1 in vascular smooth muscle cells provides a therapeutic target for the prevention of AAA formation.

Human paraoxonase gene cluster overexpression alleviates angiotensin II-induced cardiac hypertrophy in mice.

Cardiac hypertrophy is the strongest predictor of the development of heart failure, and anti-hypertrophic treatment holds the key to improving the clinical syndrome and increasing the survival rates for heart failure. The paraoxonase (PON) gene cluster (PC) protects against atherosclerosis and coronary artery diseases. However, the role of PC in the heart is largely unknown. To evaluate the roles of PC in cardiac hypertrophy, transgenic mice carrying the intact human PON1, PON2, and PON3 genes and their flanking sequences were studied. We demonstrated that the PC transgene (PC-Tg) protected mice from cardiac hypertrophy induced by Ang II; these mice had reduced heart weight/body weight ratios, decreased left ventricular wall thicknesses and increased fractional shortening compared with wild-type (WT) control. The same protective tendency was also observed with an Apoe -/- background. Mechanically, PC-Tg normalized the disequilibrium of matrix metalloproteinases (MMPs)/tissue inhibitors of MMPs (TIMPs) in hypertrophic hearts, which might contribute to the protective role of PC-Tg in cardiac fibrosis and, thus, protect against cardiac remodeling. Taken together, our results identify a novel anti-hypertrophic role for the PON gene cluster, suggesting a possible strategy for the treatment of cardiac hypertrophy through elevating the levels of the PON gene family.

High glucose concentration induces endothelial cell proliferation by regulating cyclin-D2-related miR-98.

Cyclin D2 is involved in the pathology of vascular complications of type 2 diabetes mellitus (T2DM). This study investigated the role of cyclin-D2-regulated miRNAs in endothelial cell proliferation of T2DM. Results showed that higher glucose concentration (4.5 g/l) significantly promoted the proliferation of rat aortic endothelial cells (RAOECs), and significantly increased the expression of cyclin D2 and phosphorylation of retinoblastoma 1 (p-RB1) in RAOECs compared with those under low glucose concentration. The cyclin D2-3' untranslated region is targeted by miR-98, as demonstrated by miRNA analysis software. Western blot also confirmed that cyclin D2 and p-RB1 expression was regulated by miR-98. The results indicated that miR-98 treatment can induce RAOEC apoptosis. The suppression of RAOEC growth by miR-98 might be related to regulation of Bcl-2, Bax and Caspase 9 expression. Furthermore, the expression levels of miR-98 decreased in 4.5 g/l glucose-treated cells compared with those treated by low glucose concentration. Similarly, the expression of miR-98 significantly decreased in aortas of established streptozotocin (STZ)-induced diabetic rat model compared with that in control rats; but cyclin D2 and p-RB1 levels remarkably increased in aortas of STZ-induced diabetic rats compared with those in healthy control rats. In conclusion, this study demonstrated that high glucose concentration induces cyclin D2 up-regulation and miR-98 down-regulation in the RAOECs. By regulating cyclin D2, miR-98 can inhibit human endothelial cell growth, thereby providing novel therapeutic targets for vascular complication of T2DM.

Reduced expression levels of let-7c in human breast cancer patients.

Circulating microRNAs (miRNAs) are important in the diagnosis of a number of diseases, since serum or plasma miRNAs are more stable compared with miRNA isolated from blood samples. The aim of the present study was to investigate the association between the expression levels of serum let-7c miRNA and the clinical diagnosis of breast cancer (BC). The circulating let-7c levels of 90 BC patients and 64 healthy controls were determined by performing a reverse transcription-quantitative polymerase chain reaction assay. The results demonstrated that let-7c expression was downregulated in the BC tissues compared with the paracarcinoma control tissues. In addition, the let-7c expression in the serum of BC patients was significantly lower compared with the healthy controls (P<0.01). Using a cutoff value of 0.374×103 copies/ml, the serum expression levels of let-7c exhibited 87.5% sensitivity and 78.9% specificity for distinguishing BC patients from healthy controls (area under the receiver operating characteristic curve, 0.848; 95% confidence interval, 0.785-0.911). Furthermore, the results demonstrated that the serum expression levels of let-7c were significantly higher in premenopausal compared with postmenopausal patients (P<0.05), supporting the hypothesis that postmenopausal status may affect the serum expression levels of let-7c. However, no statistically significant differences were detected in the serum levels of let-7c between ER (or PR)-positive and -negative patients. Therefore, the current study hypothesized that serum let-7c may be used as a novel and valuable biomarker for the diagnosis of BC.

Sulindac has strong antifibrotic effects by suppressing STAT3-related miR-21.

Pulmonary fibrosis (PF) is a disease with an unknown cause and a poor prognosis. In this study, we aimed to explore the pathogenesis of PF and the mechanism of sulindac in attenuating bleomycin (BLM)-induced PF. The rat PF model was induced by BLM and verified through histological studies and hydroxyproline assay. The severity of BLM-induced PF in rats and other effects, such as the extent of the wet lung to bw ratios, thickening of alveolar interval or collagen deposition, was obviously ameliorated in sulindac-treated rat lungs compared with BLM-induced lungs. Sulindac also reversed the epithelial mesenchymal transition (EMT) and inhibited the PF process by restoring the levels of E-cadherin and α-smooth muscle actin (SMA) in A549 cells. Our results further demonstrated that the above effects of sulindac might be related to regulating of interferon gamma (IFN-γ) expression, which further affects signal transducers and activators of transcription 3 (STAT3) and phosphorylated STAT3 (p-STAT3) levels. Moreover, higher miR-21 levels with the decreased E-cadherin and increased α-SMA expressions were found in transforming growth factor-ß1-treated A549 cells, which can be reversed by sulindac. Collectively, our results demonstrate that by decreasing IFN-γ-induced STAT3/p-STAT3 expression to down-regulate miR-21, sulindac could significantly reverse EMT in A549 cells and prevent BLM-induced PF.

[Intervention of Tibetan medicine--twenty wei chenxiang pill on elevation of hypoxic pulmonary arterial pressure in rats].

OBJECTIVE: To investigate the role of Tibetan medicine-Twenty Wei Chenxiang Pill interfering with serum ET-1 level, in order to confirm that ET-1 is involved to the pathogenesis of hypoxic pulmonary hypertension. METHODS: 165 Wistar rats were randomly divided into high altitude control group,Tibetan medicine-Twenty Wei Chenxiang Pill group and plain control group. The physiological signal acquisition system was used to record pulmonary arterial pressure, and RV/(LV + S) ratio were caculated. Serum HIF-1alpha and ET-1 protein levels were determined by the method of ELISA, and ETA protein levels in lung tissue were determined by Western Blot method. RESULTS: Compared with the high altitude group,in the rats of Tibetan medicine-Twenty Wei Chenxiang Pill group,the pulmonary arterial pressure decreased significantly from the seventh day and the seventh day (P < 0.01), the RV/(LV + S) ratio and serum HIF-1alpha levels decreased significantly from the third day (P < 0.05 or P < 0.01), the serum ET-1 levels decreased significantly from the third day (P < 0.05 or P < 0.01), and the expression of ETA protein decreased significantly from the beginning (P < 0.01 or P < 0.001). CONCLUSION: ET-1 is one of the important factors causing pulmonary artery pressure increasing and right ventricular wall thickening, which plays a role in hypoxic pulmonary artery only involved in the early period hypoxia, but not in the later period. Tibetan medicine--twenty Wei Chenxiang Pill can prevent the pulmonary artery hypertension and the right ventricular wall thickening in rats, and its mechanism may be related to the direct inhibition of ET-1 and protein levels of ETA or the indirect downregulation of ET-1 level and ETA through inhibition of HIF-la level.

Downregulation of ornithine decarboxylase by pcDNA-ODCr inhibits gastric cancer cell growth in vitro.

Ornithine decarboxylase (ODC), the first rate-limiting enzyme of polyamine biosynthesis, was found to be associated with cell growth, proliferation and transformation. ODC gene expression in gastric cancer was increased and its level was positively correlated with the degree of malignity of gastric mucosa and development of gastric lesions. In order to evaluate the therapeutic effects of antisense RNA of ODC on gastric cancer, an antisense RNA of ODC expressing plasmid pcDNA-ODCr which delivered a 120 bp fragment complementary to the initiation codon of ODC gene was constructed and transfected to gastric cancer cells SGC7901 and MGC803. Expression of ODC in gastric cancer cells was determined by western blot. Cell proliferation was assessed by MTS assay. Cell cycle was analyzed by flow cytometry and Matrigel assay was performed to assess the ability of gastric cancer cell invasiveness. The results showed that the ODC gene expression in gastric cancer cells transfected with the pcDNA-ODCr was downregulated efficiently. Tumor cell proliferation was suppressed significantly, and cell cycle was arrested at G1 phase. Gastric cancer cells had reduced invasiveness after gene transfer. Our study suggested that antisense RNA of ODC expressing plasmid pcDNA-ODCr had antitumor activity by inhibiting the expression of ODC, and downregulation of ODC expression using a gene therapy approach might be a novel therapeutic strategy for gastric cancer.

The expression of tumstatin is down-regulated in renal carcinoma.

Tumstatin is the 28 kDa NC1 domain of the alpha3 chain of type IV collagen that inhibits pathological angiogenesis and suppresses endothelial cell proliferation and tumor growth. In the present paper, we expressed and purified recombinant human tumstatin protein and then prepared the anti-tumstatin polyclonal antibody. To investigate the expression of tumstatin in renal carcinoma, tumstatin protein was detected by western blotting using the prepared anti-tumstatin antibody and tumstatin mRNA levels were assayed by RT-PCR. The results showed that the expression of tumstatin gene was down-regulated in renal carcinoma tissues and cells. Our study suggests that as a novel endogenous angiogenesis inhibitor, tumstatin gene expression may be a marker for diagnosis, therapy and prognosis of renal carcinoma.

Targeted antitumor effect induced by hTERT promoter mediated ODC antisense adenovirus.

The expression of Ornithine decarboxylase (ODC) which is the first key enzyme of polyamine biosynthesis is increased in cancer cells. We had blocked the polyamine synthesis pathway using the adenoviral-mediated antisense ODC in some cancer cells such as prostate cancers and colorectal cancers. These researches demonstrated that ODC antisense expression could inhibit tumor cell growth. In order to reach the goal of applying the targeting gene therapy in clinical practice, we cloned the antisense ODC RNA which was driven by cancer specific promoter (hTERT promoter; telomerase reverse transcriptase promoter) into the adenovirus vector (rAd-CMV-GFP-hTERTp-ODC). Human cancer cell lines (HepG2, Bel-7402, A549) and normal cell lines (HELF, LO2) were infected separately with rAd-CMV-GFP-hTERTp-ODC as well as with control vector (rAd-CMV-GFP). Luciferase activity assay was performed to determine hTERT promoter activity. Cell growth curves analysis, western blot analysis, flow cytometry analysis and Matrigel invasion assays were performed to assess properties of cell growth and invasiveness. The results showed that there was significant inhibition of ODC expression and cell proliferation in cancer cells treated with rAd-CMV-GFP-hTERTp-ODC compared with cells treated with PBS or rAd-CMV-GFP, and no significant inhibition was detected in normal cells. Our research offers a powerful and safe new therapeutic strategy for cancer targeted treatment.

Adenovirus vector-mediated upregulation of spermidine /spermine N1-acetyltransferase impairs human gastric cancer growth in vitro and in vivo.

Spermidine/spermine N(1)-acetyltransferase (SSAT) is the rate-limiting step in polyamine catabolism. In a previous study, we constructed a recombinant adenovirus, Ad-SSAT, which can express human SSAT. In the present study, we investigated the effect of upregulated and downregulated SSAT on gastric cancer cells. We found that upregulated SSAT could inhibit the growth of MGC803 and SGC7901 cells, whereas adverse results were found with downregulated SSAT. We further analyzed cell cycle profiles and the expression levels of the major cell cycle regulatory proteins of S phase. The results showed that the growth inhibition was caused by S phase arrest. Ad-SSAT suppressed the expression of cyclin A and nuclear factor E2F1 in MGC803 and SGC7901 cells. We observed the E2F promoter activity caused by Ad-SSAT using a reporter gene assay. We also investigated the antitumorigenicity of upregulated SSAT by Ad-SSAT using a SGC7901 xenograft model in nude mice. Our results suggest that the upregulation of SSAT by Ad-SSAT infection inhibited the growth of gastric cancer in vitro and in vivo. Ad-SSAT arrested gastric cancer cells in S phase, which was mediated through downregulation of the cyclin A-E2F signaling pathway.

Adenovirus-mediated expression of spermidine/spermine N1-acetyltransferase gene induces S-phase arrest in human colorectal cancer cells.

Spermidine/spermine N1-acetyltransferase (SSAT) is a key enzyme of polyamine catabolism. In a previous study, we constructed a recombinant adenovirus, Ad-SSAT, which can express human SSAT. In the present study, we investigated the effect of Ad-SSAT on the growth and cell cycle of colorectal cancer cells. We found that Ad-SSAT increased the expression of SSAT and inhibited the growth of HT-29 and Lovo cells. The growth inhibition was caused by cell cycle arrest in the S phase. Furthermore, Ad-SSAT was shown to suppress the expression of cyclin A and nuclear factor E2F-1 in HT-29 and Lovo cells. The inhibitory effect of Ad-SSAT on cyclin A promoter activity was also observed in a reporter gene assay. Our results suggest that the expression of SSAT mediated by Ad-SSAT infection inhibits the growth of colorectal cancer cells and induces cell cycle arrest at the S phase, through a mechanism involving the suppression of cyclin A and E2F-1 expression.

Adenovirus-mediated expression of SSAT inhibits colorectal cancer cell growth in vitro.

AIM: To construct a recombinant adenovirus that can express human spermidine/ spermine N1-acetyltransferase (SSAT) and detect its inhibitory effect on colorectal cancer cell growth in vitro. METHODS: A 516 bp cDNA of SSAT was amplified and cloned into a pGL3-hTERT plasmid. The pGL3-hTERT-SSAT recombinant was digested, and the small fragment was cloned into the shuttle vector pAdTrack. The pAdTrack-hTERT-SSAT plasmids were recombined with pAdEasy-1 vectors in AdEasy-1 cells. Positive clones were selected and transfected into the HEK293 packaging cells (transformed human embryonic kidney cells) after they were linearized by PacI. The process of adenovirus packaging and amplification was monitored by green fluorescent protein (GFP) expression. The SSAT protein levels were determined by Western blotting, and the intracellular polyamine content was detected by reverse-phase high performance liquid chromatography. The MTS (3-(4, 5-dimethylthiaol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(-4- sulfophenyl)-2H-tetrazolium, inner salt) and colony-forming assays were used to analyze the gene transduction efficiency and effect on the growth of HT-29 and LoVo cells. A viable cell count was used to determine the cell growth with or without exogenous polyamines. RESULTS: The GFP expression in 293 cells during virus packing and amplification was observed by fluorescence microscopy. Western blotting results demonstrated that Ad-hTERT-SSAT could increase the expression of SSAT, and consequently, spermidine and spermine were reduced to low levels. The MTS and colony-forming assay results showed that HT-29 and LoVo cell growth were significantly inhibited, and the inhibitory effect could be partially reversed by exogenous spermidine and spermine. CONCLUSION: The successfully constructed recombinant adenovirus Ad-hTERT-SSAT could accelerate polyamine catabolism and inhibit the colorectal cell growth in vitro. It also has therapeutic potential in the treatment of colorectal cancer.