Colon Carcinoma Cell Lines SW620 Research Articles

Effect of Bioreactor Cultures on the Proliferation and Biological Activity of Protocorm-like Bodies of Dendrobium loddigesii

Dendrobium loddigesii has long been used in traditional folk medicine. The purpose of this study was to optimize the culture conditions for its protocorm-like bodies (PLBs) and explore their biological activities. The use of an air-lift bioreactor demonstrated superior PLB proliferation compared to agitated and solid culture methods. The optimal inoculum quantity of 30 g/vessel, cultured for 28 days in the bioreactor, yielded the highest PLB biomass production. Analysis of PLB extracts revealed that the ethyl acetate (EtOAc) extract exhibited the highest levels of flavonoids and alkaloids, as well as potent antioxidant activity demonstrated by DPPH free radical scavenging assay and reducing power. Furthermore, the antiproliferative effects of the PLB extracts were assessed using MTT assays, and the EtOAc extract showed significant efficacy by reducing cell viability by over 60% in the human colon carcinoma cell line SW620 at the highest tested concentration (200 μg/mL). Mechanistic analysis revealed the downregulation of key regulatory apoptosis genes, including survivin, p53, caspase-3, and caspase-9. These results demonstrate the potential of the bioreactor culture method for the efficient production of D. loddigesii PLBs and the biological activities of the EtOAc extract, suggesting its therapeutic potential.

Annonaceous acetogenin mimic AA005 suppresses human colon cancer cell growth in vivo through downregulation of Mcl-1.

Annonaceous acetogenins are a well-established family of natural products with significant bioactivities, especially high cytotoxic and antitumor activities. AA005 is an annonaceous acetogenin mimic that has shown significant cytotoxicity against a variety of cancer cell lines, but its in vivo antitumor effects have not been demonstrated so far, and its anticancer mechanisms remain ambiguous. In this study, we investigated the effects of AA005 on human colon cancer cell lines in vivo. Human colon carcinoma cell line SW620 xenograft nude mice were treated with AA005 (5 mg/kg/day, i.p.) for 21 days. AA005 administration markedly inhibited the tumor growth via promoting nuclear translocation of apoptosis-inducing factor (AIF) and inducing AIF-dependent cell death. Subsequent studies in human colon carcinoma cell lines SW620 and RKO in vitro revealed that after the colon cancer cells exposed to AA005, downregulation of a B-cell lymphoma 2 family protein, myeloid cell leukemia-1 (Mcl-1), was an early event due to the inhibition of Mcl-1 mRNA level and protein synthesis in a time-dependent manner. Intriguingly, knockdown of Mcl-1 using small interfering RNA markedly accelerated the nuclear translocation of AIF and upregulation of receptor interacting protein-1, and enhanced AA005-mediated lethality, whereas ectopic expression of Mcl-1 substantially attenuated AA005-mediated lethality in the colon cancer cells. Finally, silencing Mcl-1 expression markedly enhanced AA005-induced lethality in SW620 xenograft nude mice, demonstrating a pivotal role of Mcl-1 downregulation in mediating the in vivo antitumor effects of AA005. Taken together, this study demonstrates for the first time the anticancer effects of AA005 against human colon cancer cell lines in vivo, which is mediated through the downregulation of Mcl-1.

Role and mechanism of Annexin A9 in the proliferation of human colon carcinoma cell line SW620

Objective To investigate the role of Annexin A9 (ANXA9) in the proliferation of human colon cancer cell line SW620 and the underlying mechanism. Methods The expression of ANXA9 in human colon cancer cell lines HT29, SW620, SW480, LoVo and SW1116 was detected by real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting. SW620 cell lines with the strongest expression of ANXA9 was transfected with 40 nmol/L ANXA9-small interfering RNA (siRNA) to inhibit the expression of ANXA9. Cell counting kit-8 (CCK-8) assay and flow cytometry (FCM) were correspondingly used to examine the effect of transfection on the activity and cell cycle of SW620 cells. The expression of proliferating cell nuclear antigen (PCNA), Cyclin D, Cyclin E, p21 and p16 was detected by Real-time PCR and Western blotting. Results The mRNA and protein expression levels of ANXA9 in SW620 cells were the highest in human colon cancer cell lines HT29, SW620, SW480, LoVo and SW1116 and mRNA expressions were as following: 0.027±0.004, 0.041±0.005, 0.028±0.003, 0.033±0.003, 0.025±0.005; protein expressions were as following: 0.287±0.076, 1.246±0.103, 0.326±0.040, 0.387±0.097, 0.295±0.064 (F=8.212, P=0.003; F=81.728, P=0.000). Expressions of ANXA9 mRNA and protein in transfection group, control group, blank group were (0.209±0.016, 0.317±0.104), (0.957±0.070, 0.647±0.155), (1.031±0.171, 0.727±0.145). The ANXA9 mRNA and protein expression in the transfection group was significantly lower than in the control and blank group (F=54.072, P=0.000; F=7.601, P=0.023). Results of CCK-8 showed that cell activity in transfection group, control group, blank group was as following: 24 h (0.605±0.024, 0.612±0.022, 0.628±0.027), 48 h (0.810±0.049, 1.196±0.118, 1.308±0.070), 72 h (1.145±0.070, 1.531±0.062, 1.454±0.081). Cell activity in transfection group were significantly lower than the control and blank group (F=38.742, P=0.000; F=32.56, P=0.000). The percentage of cells in G0/G1 phase, G2/M phase, S phase was (66.870±3.850)%, (15.060±2.480)%, (18.080±1.420)% in transfection group; 50.070±3.370)%, (32.520±2.500)%, (17.410±0.920)% in control group; (48.740±2.170)%, (32.390±1.820)%, (19.210±0.390)% in blank group. The percentage of G0/G1 phase cells in the transfection group increased (F=29.752, P=0.001), and the proportion of G2/M phase decreased (F=57.860, P=0.000), while the proportion of S phase cells had no significant change (F=1.568, P=0.284). The expression of Cyclin D1 and Cyclin E1 was significantly lower after ANXA9-siRNA transfection (mRNA: F=462.821, 5.512, P=0.000, 0.044; protein: F=15.148, 8.300, P=0.005, 0.019), and that of p21 was significantly increased (mRNA: F=6.742, P=0.032; protein: (F=21.230, P=0.002). Conclusion ANXA9 may participate in the proliferation of colon cancer cell line SW620 possibly through the expression of Cyclin D1, Cyclin E1, p21. Key words: Colon cancer; Annexin A9; Proliferation

Radio sensitization of Colon Cancer Cells Mediated by Gemcitabine through Cell Cycle Synchronization

We evaluated gemcitabine together with ionizing radiation for improved cell growth inhibition with respect to that by radiation alone in the human colon carcinoma cell lines SW620, HT-29 and SNU-C4. To this end, cells were exposed for 24 h to gemcitabine and then assessed for growth response with the sulphorhodamine B assay. The cell lines, as well as exposed to ionizing radiation and a combination of gemcitabine and ionizing radiation for 24 h, 48 h and 72 h and the radiosensitivity was assessed using a clonogenic assay. Multiple drug effect analysis was used to evaluate the synergistic effect, which was then related to the cell cycle phase distribution. The SNU-C4 cell line showed a greater sensitivity to gemcitabine in comparison to the other two cell lines, while the SW620 cells was more sensitive to damage induced by radiation. Furthermore, gemcitabine increased by 50% the effect of ionizing radiation after 24 h in SW620 cell line, while in the others cell lines, this effect was observed only after 72 h. Moreover, gemcitabine associated with ionizing radiation was synergistic in SW620, HT-29 and SNU-C4 cells. Increased in S phase fraction was seen in gemcitabine treatment in all cell lines studied. While, ionizing radiation only induced an accumulation on G2/M in SW620 and HT-29 cell lines, indicating that SNU-C4 is less sensitive to radiation effect. A significant accumulation of cells in S phase after treatment with gemcitabine followed by radiation was observed in all cell lines. In summary our data indicate that gemcitabine increases the radiosensitivity to radiation in cell lines derived from human colon cancer, and that this effect seems to be associated with the ability of gemcitabine to synchronize cells in S phase of the cell cycle.

Phosphorylation and mRNA Splicing of Collapsin Response Mediator Protein-2 Determine Inhibition of Rho-associated Protein Kinase (ROCK) II Function in Carcinoma Cell Migration and Invasion

The Rho-associated protein kinases (ROCK I and II) are central regulators of important cellular processes such as migration and invasion downstream of the GTP-Rho. Recently, we reported collapsin response mediator protein (CRMP)-2 as an endogenous ROCK II inhibitor. To reveal how the CRMP-2-ROCK II interaction is controlled, we further mapped the ROCK II interaction site of CRMP-2 and examined whether phosphorylation states of CRMP-2 affected the interaction. Here, we show that an N-terminal fragment of the long CRMP-2 splice variant (CRMP-2L) alone binds ROCK II and inhibits colon carcinoma cell migration and invasion. Furthermore, the interaction of CRMP-2 and ROCK II is partially regulated by glycogen synthase kinase (GSK)-3 phosphorylation of CRMP-2, downstream of PI3K. Inhibition of PI3K reduced interaction of CRMP-2 with ROCK II, an effect rescued by simultaneous inhibition of GSK3. Inhibition of PI3K also reduced colocalization of ROCK II and CRMP-2 at the cell periphery in human breast carcinoma cells. Mimicking GSK3 phosphorylation of CRMP-2 significantly reduced CRMP-2 binding of recombinant full-length and catalytic domain of ROCK II. These data implicate GSK3 in the regulation of ROCK II-CRMP-2 interactions. Using phosphorylation-mimetic and -resistant CRMP-2L constructs, it was revealed that phosphorylation of CRMP-2L negatively regulates its inhibitory function in ROCK-dependent haptotactic cell migration, as well as invasion of human colon carcinoma cells. Collectively, the presented data show that CRMP-2-dependent regulation of ROCK II activity is mediated through interaction of the CRMP-2L N terminus with the ROCK II catalytic domain as well as by GSK3-dependent phosphorylation of CRMP-2.

Abstract 3205: In vivo oncology target screening using a lentiviral inducible- shRNA knockdown system.

Abstract Background: Small RNAs are able to suppress expression of any gene through the endogenous RNA interference pathway and have been used to identify therapeutic targets in a variety of pathological conditions. In this project, we aimed to optimise a lentiviral inducible-knockdown small hairpin RNA (shRNA) system for efficient in vivo cancer target validation, using polo-like kinase 1 (PLK1), an essential cell-cycle protein, as a proof-of-concept target. Methods: An inducible shRNA construct targeting PLK1 or a control construct was transduced into the colon carcinoma cell line SW620. Inducible shRNA expression (marked by turbo-RFP) was monitored by fluorescence microscopy and flow cytometry in vitro and by spectrum CT in vivo. PLK1 gene expression was quantified by real-time PCR and protein expression was quantified by Western blotting in vitro or IHC ex vivo. Cell viability was assessed by MTT assays. Xenografts were established by subcutaneous injection of PLK1 inducible-knockdown cells into nude mice. Growth inhibitory effect was assessed by calliper measurement of tumour size and correlated with ex vivo assessment of gene and protein knockdown, and of cell proliferation as measured by Ki67 staining. Results: Cell viability assays demonstrated that concentrations of 2ug/ml of doxycycline or less did not result in cell death over an assay period of 7 days in both wild-type cells and cells transduced with a control shRNA construct. Following doxycycline treatment, PLK1 inducible-knockdown cells showed dose- and time-dependent shRNA induction with an increase in the level of shRNA expression and the percentage of positive cells. PLK1 down-regulation correlated with induction of shRNA expression reaching 80% gene knock-down and 60% protein knock-down 72 hours post-induction. The optimal delivery route of doxycycline via measurement of turbo-RFP induction and PK/PD measurements was assessed in vivo. In tumour-bearing mice treated with doxycycline, there was a decline in tumour growth rate in the PLK1 knockdown group compared with the control group. Ex vivo analysis showed significantly lower PLK1 gene and protein expression in the doxycycline-treated group compared with the control group which correlated with lower rates of cell proliferation in the treated group. Conclusion: The results support the anti-tumour effects of PLK1 down-regulation and confirm an efficient target validation methodology for cancer target screening using a lentiviral inducible-knockdown shRNA system. Citation Format: Yinfei Yin, Rajendra Kumari, Siddharth Subramaniam, Peter King, Sue Watson, Philip Mallinder, Anna Grabowska. In vivo oncology target screening using a lentiviral inducible- shRNA knockdown system. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3205. doi:10.1158/1538-7445.AM2013-3205

Effects on proliferation and migration of the human colon carcinoma cell line SW620 by silencing of hepatocyte growth factor expression

OBJECTIVE Hepatocyte growth factor (HGF) expression is closely related to the progression and poor prognosis of colorectal cancer patients. In this study, we investigated the effects on proliferation and migration of the human colon carcinoma cell line SW620 by silencing HGF expression. METHODS HGF was silenced using specifi c HGF α/β siRNA. The proliferation, migration, cell cycle and ultrastructure of SW620 cells were examined. RESULTS The transfection efficiency was 70%–80%. The expression rate of HGF in the experimental group was significantly lower than that in the negative and blank control groups ( P < 0.05). The proliferation inhibition rate in the experimental group at 24, 48, 72 and 96 h after transfection was 14.2%, 50.2%, 39.5% and 23.2%, respectively. The migratory ability of cells in the experimental group was significantly inhibited compared with that in the negative control or blank control groups (58.2% vs. 2.1% or 0%, P < 0.05). CONCLUSION The application of RNA interference to silence the expression of HGF in the colon carcinoma cell line SW620 actively inhibits the proliferation and migration of tumor cells.

Abstract 752: In vivo pharmaceutical targets screening using lentiviral inducible-knockdown shRNA system

Abstract Background and Aims: Small interfering RNAs (shRNAs) are able to suppress gene expression through the endogenous cellular process of RNA interference pathway. Given the ability to knock-down essentially any gene, shRNAs have been used in many studies to screen for therapeutic targets in various pathological conditions. However, most of these studies have been carried out in vitro and the results cannot always be translated into an in vivo environment. In this project, we aimed to develop an efficient in vivo cancer target validation method using a lentiviral inducible-knockdown shRNA. To verify our approach, we used an essential cell-cycle protein polo-like kinase 1 (PLK1) as a proof of concept target. Methods: TRIPZ inducible shRNAmir construct (Open Biosystems) carrying shRNA against PLK1 was transfected into the colon carcinoma cell line SW620 using lentiviral transduction. PLK1 gene knock-down following induction with doxycycline was quantified by real-time PCR. Inducible shRNAmir expression (detected by turboRFP) was monitored by fluorescence microscopy and quantified using Flow-cytometry or a fluorescence microplate reader. Nude mouse xenografts were established by subcutaneous injection of 5×106 PLK1 shRNA transfected SW620 cells into the left flank of female nude mice. Doxycycline treatment (80mg/kilogram daily via oral gavage) was initiated when the mean tumour volume reached 500mm3. Mice were sacrificed 3 weeks following treatment and tumours were collected. PLK1 gene expression was assessed by RT-PCR and protein levels investigated using immunohistochemistry. Assessment of angiogenesis (micro-vessel density) was carried out using CD34 staining. Results: Following doxycycline induction in vitro, PLK1 inducible-knockdown SW620 cells showed dose and time-dependent PLK1 down-regulation, which was consistent with shRNAmir expression as marked by turboRFP. Flow-cytometry analysis showed about 95% of cells expressed the shRNAmir, 72 hours after induction. In the in vivo model, the doxycycline-treated group showed significantly lower PLK1 gene expression compared with untreated control (60% reduction, p&amp;lt;0.003). The doxycycline-treated group also showed significantly lower PLK1 protein expression compared with the untreated control (p&amp;lt;0.001). There was a decline in tumour growth rate in the treated group compared with the control group, which confirmed that knock-down of PLK1 slowed down tumour growth. We also observed that, within the tumours from the doxycycline-treated group, there appeared to be reduced PLK1 expression in areas adjacent to the blood vessels compared with other areas of the tumour. Conclusion: The results support the anti-tumour effects of PLK1 down-regulation and confirm an efficient methodology for cancer target screening using a lentiviral inducible-knockdown shRNA system. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 752.

Protein Interaction Analysis of ST14 Domains and Their Point and Deletion Mutants

ST14 (suppression of tumorigenicity 14) is a transmembrane serine protease that contains a serine protease catalytic (SP) domain, an SEA domain, two complement subcomponent C1r/s (CUB) domains, and four low density lipoprotein receptor class A domains. Glutathione S-transferase fusion proteins with SP, CUB, and low density lipoprotein receptor domains and their corresponding mutants were generated to analyze protein interactions with these domains. Modified glutathione S-transferase pull-down assays demonstrated the interaction between the SP domain and hepatocyte growth factor activator inhibitor-1. With the same method, a CUB domain-interacting protein was isolated and turned out to be the transmembrane protein with epidermal growth factor-like and two follistatin-like domains 1 (TMEFF1). Quantitative real time PCR revealed that the expression of the TMEFF1 gene was dependent on the transfection of the ST14 gene in the RKO cell line. Our results also suggested that ST14 and TMEFF1 were co-expressed in the human breast cancer cell line MCF7, human placenta, kidney, and liver tissues. Interestingly, these two genes were co-up-regulated in kidney tumors versus normal tissues, consistent with our results that showed the dependence of TMEFF1 expression on ST14 in RKO cells. Finally, homology modeling studies suggested that TMEFF1 might form a complex with ST14 by an interaction between epidermal growth factor and CUB domains.

Sequence-dependent growth inhibition and DNA damage formation by the irinotecan–5-fluorouracil combination in human colon carcinoma cell lines

We evaluated irinotecan (CPT-11) together with 5-fluorouracil (5-FU) for improved cell growth inhibition with respect to that by either agent alone in the human colon carcinoma cell lines SW620, HT-29 and SNU-C4. Cells were exposed for 24h to each drug, as well as to various combinations and sequences of low, fixed doses of one drug with higher varying doses of the other, cultured for two more days in drug-free medium and then assessed for growth response with the sulphorhodamine B assay. Multiple drug effect analysis was used to evaluate the data, which were then related to the amount of DNA damage occurring in the cells which was determined by a fluorescence-enhancement assay for DNA unwinding. Cellular responses were also related to thymidylate synthase topoisomerase I and carboxyl esterase activities, which were assessed by a ligand-binding and a 3H-release assay; a DNA decatenation assay; and a spectrophotometric method, respectively. IC50 values for 5-FU alone in the SW620, HT29 and SNU-C4 cells were 15.3±0.8, 8.2±1.3 and 2.2±0.7μM, respectively, and for CPT-11 2.0±0.9, 2.5±0.5 and 3.8±0.3μM, respectively. The differential responses to 5-FU alone were possibly determined by differences in substrate affinity and conversion rate of thymidylate synthase (Km of approximately 7.5, 5.0 and 2.5μM and V0 of approximately 800, 200 and 2400μM/h, respectively). The comparable cellular responses to CPT-11 alone might be accounted for by the counterbalancing effects of differences in topoisomerase I (1, 1, and 1.5 arbitrary units, respectively) and carboxyl esterase activities (5055±1789, 4080±752, 1713±522mU/mg, respectively). IC20 CPT-11 prior to 5-FU was additive to synergistic in SW620, HT-29 and SNU-C4 cells (CIs of 0.7±0.1). By contrast, pretreatment with IC20 5-FU antagonised the CPT-11-mediated growth inhibition (CIs of 1.9±0.4, 1.7±1.1, 2.5±0.9, respectively). Simultaneous drug treatment did not produce more cell growth inhibition than either drug alone in the SW620 and the HT-29 cells, but was additive or antagonistic in the SNU-C4 cells (CIs of 1.1±0.3 and 2.2±1.4), depending on the ratio of the drugs. Increased DNA damage in the SW620 and HT-29 cells was only seen when IC20 CPT-11 preceded IC50 5-FU, resulting in approximately 40 and 25%, respectively, more lesions than for IC50 5-FU alone. In the SNU-C4 cells, not only such a treatment, but also simultaneous drug treatment produced (30 to 60%) more DNA damage than either drug alone. Our results show clear sequence-dependent antiproliferative effects and DNA damage formation by CPT-11 and 5-FU at combinations of low, fixed doses with higher, varying doses in cultured human colon carcinoma cells, and may be of relevance to the design of improved chemotherapeutic regimens in this disease.

TPA Induces Transglutaminase C and Inhibits Cell Growth in the Colon Carcinoma Cell Line SW620

In contrast to most other systems, TPA induced TGCactivity and protein in SW620 human colon carcinoma cells. This induction was accompanied by cell growth inhibition and increased apoptosis. The general protein kinase-C inhibitor GF-109203X blocked the induction of TGCby TPA, whereas the specific inhibitor of the PKCα isoform, the indocarbazole Go6976, reduced it by 40%. These PKC inhibitors had similar inhibitory effects on TPA increased apoptosis and inhibition of cell growth, suggesting that the observed actions of TPA are mediated by PKC, and a close connection between TGCactivity, increased apoptosis and cell growth inhibition. We conclude that TPA may offer new approaches in the management of colon cancer cell growth.

Low external pH and osmotic shock increase the expression of human MDR protein.

We have studied the effects of extracellular pH (pHo) and osmotic strength on the expression of the human multidrug resistance (MDR) protein. Both lowered pHo and hypertonic shock increase the level of hu MDR protein 5-10-fold in membranes isolated from the human colon carcinoma cell lines SW620 and HCT15 and the human kidney carcinoma line SKRC-39. Increased protein expression is dependent on the duration of acid or osmotic shock and is reversed within several days when normal growth conditions are restored. Quantitative northern blot analysis with a hu MDR 1 specific probe reveals increased MDR mRNA in the acid and hypertonically shocked cells. Interestingly, we find a greater increase in mRNA levels for hypotonically shocked colon cells, without an apparent increase in protein levels. Overexpressing cells are found to retain less [3H]vinblastine relative to cells cultured under normal conditions, and they are resistant to the cytotoxic effects of doxorubicin, vinblastine, and colchicine, but not methotrexate. This resistance appears to be reversed by treatment with verapamil. In contrast, SW620 cells previously induced to overexpress MDR protein via the administration of differentiation agents [Mickley et al. (1989) J. Biol. Chem. 264, 18031-18040] did not exhibit decreased retention of [3H]vinblastine; thus low-pHo-induced overexpression of MDR protein in these cells may provide additional factors that promote the full expression of the MDR phenotype. These data may help to explain why many solid tumors (e.g., of colon and kidney origin) develop MDR prior to chemotherapy, since they usually grow under similar acidic conditions. These data also support the contention that MDR protein may play a role in intracellular pH and volume homeostasis.