Apoptosis In SW480 Cells Research Articles

Daphnane Diterpenoids from Daphne genkwa Inhibit PI3K/Akt/mTOR Signaling and Induce Cell Cycle Arrest and Apoptosis in Human Colon Cancer Cells.

Seven new daphnane-type diterpenoids, daphgenkins A-G (1-7), and 15 known analogues (8-22) were isolated from the flower buds of Daphne genkwa. Their structures and absolute configurations were elucidated by spectroscopic data and calculated ECD analyses. The cytotoxicities of all daphnane-type diterpenoids (1-22) obtained were evaluated against three human colon cancer cell lines (SW620, RKO, and LoVo). Compounds 1, 12, and 13 exhibited cytotoxic effects against the SW620 and RKO cell lines, with IC50 values in the range of 3.0-9.7 μM. The most active new compound, 1, with an IC50 value of 3.0 μM against SW620 cells, was evaluated further for its underlying molecular mechanism. Compound 1 induced G0/G1 cell cycle arrest, leading to the induction of apoptosis in SW620 cells. Also, it induced cancer cell apoptosis by an increased ratio of Bax/Bcl-2, activated cleaved caspase-3 and caspase-9, and upregulated PARP. Finally, compound 1 significantly inhibited PI3K/Akt/mTOR signaling in SW620 cells. Together, the results suggest that compound 1 may be a suitable lead compound for further biological evaluation.

Berberine inhibits proliferation and migration of colorectal cancer cells by downregulation of GRP78.

Human colorectal cancer (CRC), a highly malignant and metastatic carcinoma, is resistant to many present anticancer therapies. The inhibition of tumor survival and growth through receptor suppression is a promising way to treat CRC. The study aimed to investigate the effect of a natural plant triterpenoid, berberine (BBR), on SW480 cells and whether its role is mediated by Glucose-regulated protein 78 (GRP78). MTT assay, wound healing assay, and Annexin V-FITC assay were used to measure the effect of BBR on the proliferation, migration, and apoptosis of SW480 cells, respectively. Immunofluorescence and western blotting were used to evaluate both the downregulation of BBR on GRP78 and the role of GRP78 in the effect of BBR on SW480 cells. Our results revealed that BBR inhibited the proliferation and migration, as well as induced the apoptosis of SW480 cells, in a dose-dependent manner. BBR induced the dose-dependent inhibition of cell proliferation in HT-29 cells. BBR inhibited the expression of GRP78 and its localization on the cell surface. Moreover, BBR inhibited the expression of Bax, Bcl-2, c-Myc, and Vimentin and up-regulated the cytokeratin expression in SW480 cells. In addition, we found that the effects of BBR on cell proliferation, migration, and apoptosis in SW480 cells were reversed by the overexpression of GRP78. Our findings demonstrated that BBR inhibited the proliferation and migration and induced the apoptosis of SW480 cells by downregulating the expression of GRP78, and targeting GRP78 might be a potential way to develop the effective anticancer therapy.

Clostridium difficile toxin B-induced apoptosis of colon cancer cells and related mechanisms

Objective To analyze the effects of Clostridium difficile toxin B (TcdB) on the proliferation and apoptosis of colon cancer cell line SW480 and the possible mechanisms related to cell apoptosis. Methods SW480 cells were treated with different concentrations of TcdB. Cell proliferation was detected by MTT assay. Cell apoptosis and mitochondrial membrane potential were measured with flow cytometry. Results TcdB significantly inhibited the proliferation of SW480 cells in a time-concentration dependent manner and the inhibition rate reached 46.36% at 48 h. Flow cytometry results showed that TcdB could induce the apoptosis of SW480 cells in a time-concentration dependent manner and a 20.83% apoptosis rate was induced by 800 ng/ml of TcdB at 48 h. Conclusions TcdB could inhibit the proliferation and induce the apoptosis of colon cancer SW480 cells, and the possible mechanisms might be relate to the initiation of mitochondrial apoptosis pathway. Key words: Clostridium difficile toxin B; Colon cancer SW480 cells; Proliferation; Apoptosis

Hypermethylation of BMP1 Promoter Regulates Migration of Colon Cancer Cells by Regulating the TGF-Beta 1/Smad2 Signaling Pathway

The infiltration and migration of colorectal cancer (CRC) cells are important factors that affect the mortality rate of CRC patients. It is essential to explore the role and mechanism of metastasis of colon cancer in clinical treatment. In this study, we show that the DNA transferase inhibitor, 5-AZA-CdR (Decitabine), affected the viability and proliferation of the CRC cell line SW480 cell, as was verified by MTT. Real-time quantitative fluorescent PCR (RT-qPCR) was used for assessing mRNA expression levels of BMP1, TGF-beta, and Smad2 in SW480 cells cultured with 5-AZA-CdR for 96 h. Expression levels of BMP1, TGF-beta, and Smad2 proteins were detected by western blotting (WB). After culturing with 5-AZA-CdR, apoptosis of SW480 cells was assessed by immunohistochemical assay for Bax and Bcl-2 expression. Transwell migration assay was used to assess the influence of reducing DNA methylation on the migration of SW480 cells. Following treatment with DNA transferase inhibitor, the cell number and viability of SW480 were reduced, their invasion and migration were inhibited, TGF-beta and Smad2 expression was decreased, and that of BMP-1, Bax, and Bcl-2 was increased. We concluded that the viability and migration of SW480 cells were inhibited by stimulating BMP1 expression, which also inhibited TGF-beta and Smad2 signaling pathways, and increased Bax and Bcl-2 expression to promote the apoptosis of SW480 cells.

MALAT1 inhibits the Wnt/β-catenin signaling pathway in colon cancer cells and affects cell proliferation and apoptosis.

Metastasis associated lung adenocarcinoma transcript 1 (MALAT1) is a highly conserved long noncoding RNA, which has been related to various pathological processes, including cancer. The role and mechanism of MALAT1 in colon cancer are not clear. We investigated MALAT1 expression in colon cancer tissues, the effect of MALAT1 on proliferation and apoptosis of SW480 cells, and the signaling pathway involved in the MALAT1 effects. MALAT1 expression was determined in 60 colon cancer and para-carcinoma tissues using reverse transcription polymerase chain reaction (RT-PCR). Knockdown of MALAT1 in SW480 cells was induced by small interfering RNA (siRNA), and the cells were divided into three groups: untreated control, nonsense siRNA-treated control, and MALAT1 siRNA-treated group. SW480 cell apoptosis was assessed using TUNEL assay and flow cytometry. Apoptosis-related and Wnt/β-catenin signaling pathway-related proteins were detected by Western blotting in SW480 cells. SW480 cell proliferation was assessed by CCK-8 assay. MALAT1 expression was significantly higher in colon cancer vs. para-carcinoma tissues. Knockdown of MALAT1 by siRNA increased the number of apoptotic cells and the apoptosis rate at 24 h post-transfection in SW480 cells. Bcl2 associated X protein (Bax) expression was increased, B-cell lymphoma 2 (Bcl-2) expression was decreased, and the ratio of cleaved caspase-3 to truncated caspase-3 was increased in MALAT1 siRNA-transfected SW480 cells. MALAT1 knockdown decreased the proliferation of SW480 cells at 24 h, 48 h, and 72 h post-transfection. Wnt and β-catenin expression was inhibited in MALAT1 siRNA-transfected SW480 cells. Inhibition of MALAT1 expression in colon cancer may promote apoptosis and hinder cell proliferation by suppressing the activation of Wnt/β-catenin signaling pathway.

Hsa-miR-6165 downregulates insulin-like growth factor-1 receptor (IGF-1R) expression and enhances apoptosis in SW480 cells.

MicroRNAs are small non-coding RNAs that are implicated in various biological processes. Hsa-miR-6165 (miR-6165), located in the p75NTR gene, is known to induce apoptosis in human cell lines, but its mechanism of action is not fully understood yet. Here, we predicted the insulin-like growth factor 1 receptor (IGF-1R) gene as a bona fide target for miR-6165. The overexpression of miR-6165 in SW480 cells resulted in significant downregulation of IGF-1R expression as detected by real time quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA). Also, it resulted in reduced transcript levels of AKT2, AKT3, PI3KR3, PI3KR5, CCND1, c-MYC and P21 genes detected by RT-qPCR analysis. In addition, a direct interaction between miR-6165 and a 3'UTR sequence of the IGF-1R gene was verified through a dual luciferase assay. Furthermore, miR-6165 and IGF-1R showed opposite patterns of expression during the neural differentiation process of NT2 cells. Annexin V analysis and MTT assay showed that miR-6165 overexpression was followed by increased apoptosis and reduced the viability rate of SW480 cells. Moreover, a lower expression level of miR-6165 was detected in high-grade colorectal tumors compared with low-grade tumors. Taken together, the results of our study suggest a tumor suppressive role of miR-6165 in colorectal cancer, which seems to take place by regulating IGF-1R gene expression.

Inhibitory Effects of Pectinase-Treated Prunus Mume Fruit Concentrate on Colorectal Cancer Proliferation and Angiogenesis of Endothelial Cells.

Pectinase is a well-known enzyme used in the food processing industry to produce fruit juice and concentrate. This study evaluated the anticancer and antiangiogenesis activities of pectinase-treated Prunus mume fruit concentrate (PC) and its phenolic components. PC treatment (250 to 1,000µg/mL) resulted in decreased proliferation of SW480 human colorectal cancer cells through S-phase cell cycle arrest; however, equivalent concentrations of PC did not show toxicity toward CRL-1539 colon normal cells. Furthermore, PC-induced caspase-dependent apoptosis in SW480 cells, which was characterized by accumulation of apoptotic cell population, cell shrinkage, formation of apoptotic bodies, upregulation of proapoptotic Bax, cleaved PARP, caspase-3, caspase-8, and caspase-9, and downregulation of antiapoptotic Bcl-2. Antiangiogenesis effects of PC were assessed using human umbilical vein endothelial cells (HUVECs). We found that PC did not inhibit HUVECs proliferation at concentrations of 500 to 1,500µg/mL. In addition, treatment with PC at nontoxic concentrations (500 to 1,000µg/mL) blocked vascular endothelial growth factor induced cell migration, invasion, capillary-like tube formation, and angiogenesis from rat aortic rings. HPLC-PDA analysis showed that there were at least four different phenolics including 5-HMF, neochlorogenic acid, protocatechuic acid, and syringic acid. Taken together, these results indicated that PC could be used as a good source of phenolic compounds with selective anticancer and antiangiogenesis activities. PRACTICAL APPLICATION: Pectinases are one of the well-known enzyme used in the part of food processing. Treatment of pectinase is a useful strategy to reduce viscosity, turbidity, and pulp particles in the production of fruit juice, extract, and concentrate. In the present study, we found that pectinase-treated P. mume fruit concentrate significantly suppresses colorectal cancer proliferation and angiogenesis of human umbilical vein endothelial cells. The significance of our findings is that pectinase-treated P. mume concentrate may be used as a commercial functional food material to inhibit colorectal cancer and angiogenesis.

PEGylated gold nanoparticles-ribonuclease induced oxidative stress and apoptosis in colorectal cancer cells.

Introduction: Currently, drug-induced reactive oxygen species (ROS) mediating apoptosis pathway have extensively been investigated in designing effective strategies for colorectal cancer (CRC) chemotherapy. Bovine pancreatic ribonuclease A (RNase A) represents a new class of cytotoxic and non-mutagenic enzymes, and has gained more attention as a potential anticancer modality; however, the cytosolic ribonuclease inhibitors (RIs) restrict the clinical application of this enzyme. Nowadays, nanotechnology-based diagnostic and therapeutic systems have provided potential solutions for cancer treatments. Methods: In this study, the gold nanoparticles (AuNPs) were synthesized, stabilized by polyethylene glycol (PEG), functionalized, and covalently conjugated with RNase A. The physicochemical properties of engineered nanobiomedicine (AuNPs-PEG-RNase A) were characterized by scanning electron microscope (SEM), dynamic light scattering (DLS), and UV-vis spectrum. Then, its biological impacts including cell viability, apoptosis, and ROS production were evaluated in the SW-480 cells. Results: The engineered nanobiomedicine, AuNPs-PEG-RNase A, was found to effectively induce apoptosis in SW-480 cells and result in a significant reduction in cancer cell viability. Besides, the maximum production of ROS was obtained after the treatment of cells with an IC50 dose of AuNPs-PEG-RNase A. Conclusion: Based on the efficient ROS-responsiveness and the anticancer activity of RNase A of the engineered nanomedicine, this nanoscaled biologics may be considered as a potential candidate for the treatment of CRC.

Triterpenoids Extracted from Rhus chinensis Mill Act Against Colorectal Cancer by Inhibiting Enzymes in Glycolysis and Glutaminolysis: Network Analysis and Experimental Validation

Background: Rhus chinensis Mill is a traditional Chinese medicine (TCM) mostly used to treat several cancer types. Although previous studies have found that certain ingredients of R. chinensis such as flavonoids can inhibit tumor cell proliferation [e.g. colorectal cancer (CRC)], systematic research on the mechanism underlying anticancer effect of active compounds like triterpenoids (TER) is lacking.Study Design: Herein, the concept of “network pharmacology primarily based on active compounds” was applied to explore the anticancer mechanisms of TER extract from R. chinensis. In this regard, potential targets and pathways of glycolysis and glutaminolysis form the basis for the anti-CRC effect of triterpenoids. Network pharmacology was used to predict several key proteins in the metabolic pathways, which were further verified via western blot and metabolomics methods.Results: Our results showed that the total TER in R. chinensis remarkably inhibited the proliferation and apoptosis of SW620 cells. The top 4 compounds of TER (viz., betulinic acid-BTA, betulonic acid-BTOA, betulin-BT, and semialactic acid-SA) were confirmed through the detection of UPLC-MS and analysis of cell proliferation assays. Mechanistically, this study revealed that TER plays an anti-CRC role through key targets, such as ENO1, ALDOA, PFKFB3, PKM2, and LDHA, as well as key glycolytic and glutaminolytic pathways.Conclusion: Collectively, these results have provided new insights into the mechanism underlying anti-CRC effect of triterpenoids extract obtained from R. chinensis, mainly through combination of compositional quantitative analysis, network pharmacology, and experimental verification.

Peroxiredoxin V Reduces β-Lapachone-induced Apoptosis of Colon Cancer Cells.

Peroxiredoxin (Prx) V has been known as an antioxidant enzyme which scavenges intracellular reactive oxygen species (ROS). Also, Prx V has been shown to mediate cell apoptosis in various cancers. However, the mechanism of Prx V-induced apoptosis in colon cancer cells remains unknown. Thus, in this study we analyzed the effects of Prx V in β-lapachone-induced apoptosis in SW480 human colon cancer cells. β-lapachone-induced apoptosis was analyzed by the MTT assay, western blotting, fluorescence microscopy, Annexin V staining and flow cytometry. Overexpression of Prx V, significantly decreased β-lapachone-induced cellular apoptosis and Prx V silencing increased β-lapachone-induced cellular apoptosis via modulating ROS scavenging activity compared to mock SW480 cells. In addition, to further explore the mechanism of Prx V regulated β-lapachone-induced SW480 cells apoptosis, the Wnt/β-catenin signaling was studied. The Wnt/ β-catenin signaling pathway was found to be induced by β-lapachone. Prx V regulates SW480 cell apoptosis via scavenging ROS cellular levels and mediating the Wnt/β-catenin signaling pathway, which was induced by β-lapachone.

Potential of Teucrium chamaedrys L. to modulate apoptosis and biotransformation in colorectal carcinoma cells

Ethnopharmacological relevanceTeucrum chamaedrys L. is one of the known medicinal plants, useful for treatment of various health problems, especially digestive. In this study, we investigated methanol, ethyl-acetate and acetone extracts of T. chamaedrys in respect to their anticancer properties in SW480 colorectal cancer cells. Materials and methodsCytotoxicity and proapoptotic potential were assessed by MTT cell viability assay and AO/EB double staining. Molecular mechanisms of induced apoptosis were determined by monitoring Fas receptor protein expression through immunofluorescence, Caspase 8 and 9 activity, as well as concentrations of O2.- spectrophotometrically. Additionally, mRNA expression of biotransformation enzymes (CYP1A1, CYP1B1, GSTP1) and membrane transporters (MRP1 and MRP2) involved in drug resistance were investigated by qPCR method. Qualitative analysis of individual phenolic compounds was performed by reversed phase HPLC-MS analysis. ResultsMethanol extract shows the best cytotoxicity and selectivity compared to ethyl-acetate and acetone extracts, mainly causing apoptosis of SW480 cells, without affecting normal HaCaT keratinocytes. The increased expression of Fas receptor protein and caspase 8 activity indicate that the death receptor-mediated pathway plays a crucial role in the observed apoptosis. The increased caspase 9 activity and O2.- concentration suggest that mitochondria are also involved in the apoptosis. T. chamaedrys methanol extract inhibits mRNA expression of CYP1A1, CYP1B1, GSTP1, MRP1 and MRP2 in SW480 cells. ConclusionsInduction of apoptosis and inhibition of CYP1A1, CYP1B1, GSTP1, MRP1 and MRP2 mRNA expression implies that T. chamaedrys can serve as a valuable source of bioactive compounds as dietary supplements or selective anticancer agents, with the ability to induce apoptosis and modulate drug resistance in colorectal cancer cells.

MiR-1271 enhances the sensitivity of colorectal cancer cells to cisplatin.

The high mortality of colorectal cancer (CRC) is likely caused by early invasion and metastasis. The chemoresistance of tumor cells is the critical reason for treatment failure. The present study aimed to develop targeted solutions to overcome chemotherapy drug resistance in CRC. CCK-8 assay was used to examine SW480 cell viability. SW480 cell apoptosis was examined using flow cytometry. The present study demonstrated that the expression of miR-1271 was significantly decreased in CRC tumors and cell lines compared with control tissues. Furthermore, the expression of microRNA (miR)-1271 was increased and decreased following the transfection of miR-1271 mimics and an inhibitor, respectively. Furthermore, miR-1271 regulated mammalian target of rapamycin (mTOR) expression by directly binding to the mTOR 3′-untranslated region and the relative luciferase activity of mTOR was decreased following miR-1271 overexpression. The results of the present study indicate that miR-1271 may be a potential target for anti-CRC therapy, particularly in the sensitivity of chemotherapeutic drugs. miR-1271 may therefore enhance the sensitivity of CRC cells to chemotherapy drugs and provide a novel approach for the gene therapy of CRC.

The Cellular Uptake and Apoptotic Efficiency of Colchicine is Correlated with Downregulation of MMP-9 mRNA Expression in SW480 Colon Cancer Cells.

Colchicine, a tricyclic alkaloid, is commonly used in treatment due to its antiinflammatory and anti-fibrotic effects. Besides its toxicity at high doses, colchicine is reported for its potential anticancer effects at lower concentrations. The present study aimed to evaluate the anticancer effects of colchicine in SW480 cells. The effect of colchicine on cell proliferation was determined by MTT assay. The cellular colchicine uptake was measured by HPLC analysis. The apoptotic effects was evaluated by annexin v binding assay and MMP-9 mRNA expression was determined by RT-PCR experiments. Colchicine showed significant cytotoxicity at 10 ng/ml and higher concentrations and caused a cell cycle arrest of SW480 cells at G2/M phase. The results of HPLC analysis showed that colchicine uptake was increased in correlation with treated concentrations. Colchicine concentrations have increased the amount of apoptotic cell population. The elisa and western blot measurements showed that colchicine led to nuclear translocation of NF-κB proteins and increased caspase levels. The real time PCR experiments showed that colchicine has inhibitory effect on MMP-9 mRNA expression in a concentration dependent manner. These results illustrated that low dose colchicine efficiently induced cell death and apoptosis of SW480 cells and the inhibition of MMP-9 mRNA levels was significantly correlated with the amount of cellular colchicine uptake, suggesting that colchicine has a potential value in the treatment of human colorectal cancer.

Extracts from Sageretia thea reduce cell viability through inducing cyclin D1 proteasomal degradation and HO-1 expression in human colorectal cancer cells

BackgroundSageretia thea (S. thea) has been used as the medicinal plant for treating hepatitis and fevers in Korea and China. Recently, anticancer activity of S. thea has been reported, but the potential mechanism for the anti-cancer property of S. thea is still insufficient. Thus, we evaluated whether extracts from the leaves (STL) and branches (STB) of S. thea exert anticancer activity and elucidated its potential mechanism in SW480 cells.MethodsMTT assay was performed for measuring cell viability. Western blot and RT-PCR were used for analyzing the level of protein and mRNA, respectively.ResultsTreatment of STL or STB decreased the cell viability and induced apoptosis in SW480 cells. Decreased level of cyclin D1 protein was observed in SW480 cells treated with STL or STB, but no change in cyclin D1 mRNA level was observed with the treatment of STL or STB. MG132 blocked downregulation of cyclin D1 protein by STL or STB. Thr286 phosphorylation of cyclin D1 by STL or STB occurred faster than downregulation of cyclin D1 protein in SW480 cells. When SW480 cells were transfected with T286A-cyclin D1, cyclin D1 degradation by STL or STB did not occur. Inhibition of GSK3β and cyclin D1 nuclear export attenuated STL or STB-mediated cyclin D1 degradation. In addition, STL or STB increased HO-1 expression, and the inhibition of HO-1 attenuated the induction of apoptosis by STL or STB. HO-1 expression by STL or STB resulted from Nrf2 activation through ROS-dependent p38 activation.ConclusionsThese results indicate that STL or STB may induce GSK3β-dependent cyclin D1 degradation, and increase HO-1 expression through activating Nrf2 via ROS-dependent p38 activation, which resulted in the decrease of the viability in SW480 cells. These findings suggest that STL or STB may have great potential for the development of anti-cancer drug.

Baicalin induces apoptosis in SW480 cells through downregulation of the SP1 transcription factor.

Colorectal cancer occurs throughout the world but is most common in developed countries. Cancer progression is believed to be driven by genetic mutations in this complex condition. Risk factors for developing colorectal cancer include a genetic family history, long-term ulcerative colitis, and colonic polyps. The use of baicalin has been reported to be clinically efficacious against colon tumors in Asian countries despite an unclear mechanism of action. Several cancers have been found to be biologically dependent on the specificity protein 1 (sp1) transcription factor family. We hypothesized that baicalin may exert its chemotherapeutic effects by sp1 downregulation. Using the SW480 human colorectal cancer cell line, we investigated the physiological properties of baicalin. Our experiments were designed toward clarifying three goals: (a) to determine the mRNA expression profile of transcription factors in colorectal cancer patients using a microarray-based analysis; (b) to determine the effects of baicalin on the sp1 transcription factor with western blotting and reporter cell assays; and (c) to contrast the effects of mithramycin-A (an sp1 transcription factor inhibitor) and baicalin using western blotting and reporter cell assays. Both baicalin and mithramycin-A downregulated sp1 expression, attenuated SW480 cell proliferation, and increased cell apoptosis. Baicalin inhibited sp1 expression and led to SW480 apoptosis, thus clarifying the effect of this traditional Chinese medicine compound in the treatment of colon cancer.

The combined treatment of brassinin and imatinib synergistically downregulated the expression of MMP-9 in SW480 colon cancer cells.

In cancer treatment, which is a major cause of mortality today, combination studies with clinically used chemotherapeutics are becoming increasingly important as much as investigating the effects of novel natural compounds. In this context, phytoalexins constitute an important group due to their unique structure. Brassinin is an essential indole phytoalexin and is a biosynthetic precursor for other phytoalexins. The purpose of this study was to evaluate the anticancer effects of brassinin in combination with imatinib in SW480 cells. In the study, it was observed that brassinin-imatinib combination significantly increased cytotoxicity compared with the single treatment of both compounds and inhibited cell cycle at G0/G1 phase. Annexin V binding and fluorescence imaging assays showed that the combination of brasinin-imatinib induces apoptosis in a dose-dependent manner. Furthermore, the effect of brassinin on the activity of MMP-9 in SW480 cells was evaluated for the first time, and it was detected that MMP-9 activity was significantly reduced. The combination of brassinin-imatinib was found to inhibit MMP-9 activity as well as relative MMP-9 gene expression on a higher level compared with control and compounds alone. Our findings have revealed that the combination of brassinin-imatinib synergistically induces cytotoxicity and apoptosis in SW480 cells. The findings on MMP-9 downregulation have also revealed the anti-metastatic potential of treatment.

Regulation between two alternative splicing isoforms ZNF148FL and ZNF148ΔN, and their roles in the apoptosis and invasion of colorectal cancer

ObjectiveTo investigate the effect of two alternative splicing isoforms of zinc finger protein (ZNF) 148 gene on the invasion and metastasis of human colorectal cancer (CRC) cells and their related mechanisms. MethodsQuantitative RT-PCR assays were used to detect the expression of twoZNF148 alternative splicing isoforms in SW480 cells. ZNF148FL-siRNA, ZNF148FL-over express vector, ZNF148ΔN-siRNA, and ZNF148ΔN-over express vector were introduced into SW480 cells. The transfection efficiency was confirmed by RT-PCR. The proliferation, invasion, and migration in vitro as well as the apoptosis of SW480 cells were detected by MTT, transwell, scratch assay and flow cytometry, respectively. ResultsBoth ZNF148FL and ZNF148ΔN were expressed in SW480 cells, and the level of ZNF148FL protein was higher than ZNF148ΔN. After ZNF148FL-siRNA and ZNF148ΔN-over express transfection, the expression level of ZNF148FL and ZNF148ΔN were significantly decreased and increased, respectively. In contrast, the expression of ZNF148FL and ZNF148ΔN were significantly increased and decreased, respectively, after ZNF148FL-over express and ZNF148ΔN-siRNA transfection (all P < 0.05). The proliferation of SW480 cells was increased in ZNF148FL-over express group and the ZNF148ΔN-siRNA group, while decreased in ZNF148FL-siRNA group and ZNF148ΔN-over express group. The invaded cell number and migrated distance in ZNF148FL-siRNA group and ZNF148ΔN-over express group were significantly decreased, but the apoptotic rate was significantly increased. In contrast, ZNF148FL-over express and ZNF148ΔN-siRNA group showed the significantly increased ability of invasion and migration but decreased apoptosis rate (all P < 0.05). ConclusionZNF148FL could increase proliferation, invasion, and migration of CRC cells, while ZNF148ΔN showed opposite effect; the two splicing isoforms of ZNF148 may exert a mutual antagonistic effect to each other on the malignant biological activities.

Tanshinone IIA promotes IL2-mediated SW480 colorectal cancer cell apoptosis by triggering INF2-related mitochondrial fission and activating the Mst1-Hippo pathway.

IL-2-based therapy is a promising tool to treat colorectal cancer, but drug resistance always occurs in clinical practice. Mitochondrial fission is a novel target to modulate cancer development and progression. The aim of our study is to explore the effect of IL-2 combined with Tan IIA on SW480 colorectal cancer cell apoptosis in vitro and to determine whether IL-2/Tan IIA cotreatment could reduce SW480 cell viability via activating mitochondrial fission. The results indicated that Tan IIA increased IL-2-mediated cell death in SW480 colorectal cancer cells, and this effect was also accompanied with a reduction in cell proliferation. Functional investigations demonstrated that Tan IIA/IL-2 cotreatment enhanced INF2-related mitochondrial fission. Excessive mitochondrial division induced mitochondrial oxidative stress, mitochondrial energy metabolism disorder and mitochondrial apoptosis in SW480 cells. Inhibition of mitochondrial fission attenuated the antitumor effect of Tan IIA/IL-2 cotreatment on SW480 cell apoptosis. Further, we demonstrated that Tan IIA/IL-2 combination therapy controlled INF2-related mitochondrial fission via the Mst1-Hippo pathway. Moreover, Mst1 knockdown abrogated Tan IIA/IL-2-activated mitochondrial fission. Altogether, our results demonstrated that Tan IIA enhances the therapeutic efficiency of IL-2-mediated SW480 colorectal cancer cell apoptosis via promoting INF2-related mitochondrial fission and activating the Mst1-Hippo pathway.

Effect of the combined use of oncolytic adenovirus carrying short hairpin RNA targeting special AT rich sequence binding protein-1 with L-oxaliplatin on the proliferation and apoptosis of colorectal cancer cells

Objective To observe the effects of the combined use of oncolytic adenovirus carrying short hairpin RNA (shRNA) targeting special AT rich sequence binding protein-1 (SATB1) (ZD55-SATB1) with L-oxaliplatin (L-OHP) on the proliferation and apoptosis of colorectal cancer SW620 cells. Methods SW620 cells were divided into 5 groups and treated with phosphate buffer (PBS), L-OHP (16 μg/ml), ZD55-enhanced green fluorescent protein (EGFP, 10 multiplicity of infection, MOI), ZD55-SATB1 (10 MOI), ZD55-SATB1 (10 MOI) plus L-OHP (16 μg/ml) respectively. The viability of SW620 cells treated with L-OHP (0, 4, 8, 32 μg/ml) alone and ZD55-SATB1 (0, 0.1, 1.0, 10.0, 50.0 MOI) alone for 96 h was assessed by cell counting kit-8 (CCK-8) assay. And the viability of SW620 cells of 5 groups with 48 h treatment was assessed by CCK-8 assay. Cell apoptosis was determined by Annexin V propidium iodide (PI)/fluoresceine isothiocyanate (FITC) and Hoechst 33258 assay. The expression of E1A, SATB1 and apoptosis associated protein cysteinyl aspartate-specific protease (Caspase)-3, Caspase-8, B cell lymphoma/leukemia-2 (bcl-2) and bcl-2 associated X protein (bax) in SW620 cells was detected by Western blotting. Results CCK-8 assay showed that ZD55-SATB1 (10 MOI) plus L-OHP (16 μg/ml) (35.22±2.42)%, compared with corresponding dose ZD55-SATB1 (10 MOI) (52.18±2.85)% (P=0.028), ZD55-EGFP (10 MOI) (60.19±0.82)% (P=0.036) and L-OHP (16 μg/ml) (63.29±2.01)% (P=0.014), could significantly inhibit the viability of SW620 cells. Hoechst33258 staining showed the combination of ZD55-SATB1 (10 MOI) with L-OHP (16 μg/ml) (59.49±2.53)% produced obvious apoptosis of the SW620 cells as compared with ZD55-SATB1 (10 MOI) (44.24±8.81)% (P=0.019), ZD55-EGFP (10 MOI) (23.59±6.28)% (P=0.026) and L-OHP (16 μg/ml) (8.61±2.83)% (P=0.015), and the difference was statistically significant. Annexin V assay showed that ZD55-SATB1 (10 MOI) plus L-OHP (16 μg/ml) (55.84±2.14)%, as compared with corresponding dose ZD55-SATB1 (10 MOI) (36.17±1.51)% (P=0.018), ZD55-EGFP (10 MOI) (25.43±3.25)% (P=0.012) and L-OHP (16 μg/ml) (23.86±1.92)% (P=0.017), could significantly accelerate the apoptosis of SW620 cells. Western blotting showed that the adenovirus ZD55-SATB1 and ZD55-EGFP were efficient replication in SW620 cells and L-OHP had no effect on adenovirus replication. In addition, SATB1 expression in the ZD55-SATB1 plus L-OHP group was lower than PBS group, L-OHP group, ZD55-SATB1 group and ZD55-EGFP group. Western blotting analysis also confirmed that ZD55-SATB1 (10 MOI) plus L-OHP (16 μg/ml) could increase the expression level of proapoptotic protein Caspase-3, Caspase-8, bax, concomitant with down-regulating the expression level of antiapoptotic proteins bcl-2 (P=0.000). Conclusion The combined use of ZD55-SATB1 with L-OHP could significantly inhibit the proliferation and induce proapoptotic effect in SW620 cells. And the combination of the treatment of oncolytic adenovirus and L-OHP is better than L-OHP or adenovirus alone. Key words: Special AT rich sequence binding protein-1; Colorectal cancer; Oxaliplatin; Proliferation

MicroRNA-488 inhibits progression of colorectal cancer via inhibition of the mitogen-activated protein kinase pathway by targeting claudin-2.

Colorectal cancer (CRC) affects people globally, and lymph node metastasis (LNM) is an important indicator of poor clinical outcome in CRC. The current study aims to evaluate the role of microRNA-448 (miR-488) and claudin-2 (CLDN2) in epithelial-mesenchymal transition (EMT) and LNM of CRC through the MAPK signaling pathway. First, microarray analysis indicated that miR-488 was poorly expressed in CRC, whereas CLDN2 was highly expressed. Additionally, the bioinformatics website MicroRNA.org and the dual luciferase reporter gene assay found that CLDN2 was a target gene of miR-488. Next, the results for the correlations between expression of miR-488 and clinicopathological characteristics of CRC indicated that the expression of miR-488 was closely associated with differentiation degree, LNM, and Dukes stages in CRC patients. Moreover, overexpression of miR-488 inhibited the activation of the MAPK signal transduction pathway. Notably, loss- and gain-of-function experiments demonstrated that upregulation of miR-488 suppressed SW480 cell viability, invasion, and migration and promoted apoptosis in SW480 cells. Finally, overexpression of miR-488 inhibited LNM, microlymphatic vessel density, and tumor growth in nude mice. We conclude that overexpression of miR-488 could suppress the cell proliferation, EMT, and LNM of CRC cells via inhibition of the CLDN2-mediated MAPK signaling pathway, which could be a new molecular therapy target for CRC.