• Asian Pacific Journal of Cancer Prevention
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• 제15권15호
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• pp.6321-6326
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• 2014

${\alpha}$-Methyl-n-butylshikonin (MBS), one of the active components in the root extracts of Lithospermum erythrorhizon, posses antitumor activity. In this study, we assess the molecular mechanisms of MBS in causing apoptosis of SW620 cells. MBS reduced the cell viability of SW620 cells in a dose-and time-dependent manner and induced cell apoptosis. Treatment of SW620 cells with MBS down-regulated the expression of Bcl-2 and up-regulated the expression of Bak and caused the loss of mitochondrial membrane potential. Additionally, MBS treatment led to activation of caspase-9, caspase-8 and caspase-3, and cleavage of PARP, which was abolished by pretreatment with the pan-caspase inhibitor Z-VAD-FMK. MBS also induced significant elevation in the phosphorylation of JNK and p38. Pretreatment of SW620 cells with specific inhibitors of JNK (SP600125) and p38 (SB203580) abrogated MBS-induced apoptosis. Our results demonstrated that MBS inhibited growth of colorectal cancer SW620 cells by inducing JNK and p38 signaling pathway, and provided a clue for preclinical and clinical evaluation of MBS for colorectal cancer therapy.

• Asian Pacific Journal of Cancer Prevention
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• 제14권9호
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• pp.4999-5005
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• 2013

Objective: This study aimed to investigate expression of the proto-oncogene POK erythroid myeloid ontogenic factor (Pokemon) in colorectal cancer (CRC), and assess inhibitory effects of a small interference RNA (siRNA) expression vector in SW480 and SW620 cells. Methods: Semi-quantitative reverse transcription-polymerase chain reaction (PCR) and immunohistochemistry were performed to determine mRNA and protein expression levels of Pokemon in CRC tissues. Indirect immunofluorescence staining was applied to investigate the location of Pokemon in SW480 and SW620 cells. The siRNA expression vectors that were constructed to express a short hairpin RNA against Pokemon were transfected to the SW480 and SW620 cells with a liposome. Expression levels of Pokemon mRNA and protein were examined by real-time quantitative-fluorescent PCR and western blot analysis. The effects of Pokemon silencing on proliferation of SW480 and SW620 cells were evaluated with reference to growth curves with MTT assays. Results: The mRNA expression level of Pokemon in tumor tissues ($0.845{\pm}0.344$) was significantly higher than that in adjacent tumor specimens ($0.321{\pm}0.197$). The positive expression ratio of Pokemon protein in CRC (87.0%) was significantly higher than that in the adjacent tissues (19.6%). Strong fluorescence staining of Pokemon protein was observed in the cytoplasm of the SW480 and SW620 cells. The inhibition ratios of Pokemon mRNA and protein in the SW480 cells were 83.1% and 73.5% at 48 and 72 h, respectively, compared with those of the negative control cells with the siRNA. In the SW620 cells, the inhibition ratios of Pokemon mRNA and protein were 76.3% and 68.7% at 48 and 72 h, respectively. MTT showed that Pokemon gene silencing inhibited the proliferation of SW480 and SW620 cells. Conclusion: Overexpression of Pokemon in CRC may have a function in carcinogenesis and progression. siRNA expression vectors could effectively inhibit mRNA and protein expression of Pokemon in SW480 and SW620 cells, thereby reducing malignant cell proliferation.

• 대한의생명과학회지
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• 제28권1호
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• pp.42-49
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• 2022

Tetrodotoxin (TTX) is a natural neurotoxin found in several species of puffer fish belonging to Tetraodon fugu genus and has been reported to affect processes such as proliferation, metastasis and invasion of various cancer cells. However, it was not revealed which genes were influenced by these reactions. In this experiment, it was examined in human SW620 colorectal cancer cells. The proliferation of SW620 cells was significantly reduced when treated with 0, 1, 10 and 100 μM TTX for 48 h. It was confirmed using Annexin V-propidium iodide staining that some apoptosis was induced. Differentially expressed genes (DEGs) affecting cell proliferation through RNA sequencing (RNA-seq) were selected. The expression change of DEGs was confirmed by conducting quantitative real-time polymerase chain reaction (qRT-PCR). As a result, the mRNA expression of FOS and WDR48 genes was found to be increased in the 100 μM TTX treatment group compared to the control group. On the other hand, the mRNA expression of ALKBH7, NDUFA13, RIPPLY3 and SELENOM genes was found to be reduced, and in the case of the ALKBH7 gene was identified to show significant differences. This experiment suggests that TTX can be used as an important fundamental data to elucidate the mechanism that inhibits the proliferation of SW620 cells.

• Asian Pacific Journal of Cancer Prevention
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• 제15권6호
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• pp.2911-2916
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• 2014

Oxaliplatin is a first-line therapy for colorectal cancer, but cancer cell resistance to the drug compromises its efficacy. To explore mechanisms of drug resistance, we treated colorectal cancer cells (HCT116 and SW620) long-term with oxaliplatin and established stable oxaliplatin-resistant lines (HCT116-OX and SW620-OX). Compared with parental cell lines, $IC_{50}$s for various chemotherapeutic agents (oxaliplatin, cisplatin and doxorubicin) were increased in oxaliplatin-resistant cell lines and this was accompanied by activation of nuclear factor erythroid-2 p45-related factor 2 (Nrf2) and NADPH quinone oxidoreductase 1 (NQO1). Furthermore, luteolin inhibited the Nrf2 pathway in oxaliplatin-resistant cell lines in a dose-dependent manner. Luteolin also inhibited Nrf2 target gene [NQO1, heme oxygenase-1 (HO-1) and $GST{\alpha}1/2$] expression and decreased reduced glutathione in wild type mouse small intestinal cells. There was no apparent effect in Nrf2-/- mice. Luteolin combined with other chemotherapeutics had greater anti-cancer activity in resistant cell lines (combined index values below 1), indicating a synergistic effect. Therefore, adaptive activation of Nrf2 may contribute to the development of acquired drug-resistance and luteolin could restore sensitivity of oxaliplatin-resistant cell lines to chemotherapeutic drugs. Inhibition of the Nrf2 pathway may be the mechanism for this restored therapeutic response.

• 약학회지
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• 제54권3호
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• pp.192-199
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• 2010

Colorectal cancer is one of the most common alimentary malignancies. In this study, the antitumor activity of activated human natural killer (NK) cells against human colorectal cancer was evaluated in vivo. Human NK cells are the key contributors of innate immune response and the effective functions of these cells are enhanced by cytokines. Human peripheral blood mononuclear cells (PBMC) were cultured with interleukin-2 (IL-2)-containing medium for 14 days and resulted in enriched NK cell population. The resulting populations of the cells comprised 7% $CD3^+CD4^+$ cells, 25% $CD3^+CD8^+$ cells, 13% $CD3^-CD8^+$ cells, 4% $CD3^+$CD16/$CD56^+$ cells, 39% $CD3^+$CD16/$CD56^-$ cells, and 52% $CD3^-$CD16/$CD56^+$ cells. Tumor necrosis factor alpha (TNF-$\alpha$), interferon gamma (IFN-$\gamma$), IL-2, IL-4, and IL-5 transcripts of the activated NK cells were confirmed by RT-PCR. In addition, activated NK cells at doses of 2.5, 5 and 10 million cells per mouse inhibited 10%, 34% and 47% of SW620-induced tumor growth in nude mouse xenograft assays, respectively. This study suggests that NK cell-based immunotherapy may be used as an adoptive immunotherapy for colorectal cancer patients.

• Asian Pacific Journal of Cancer Prevention
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• 제14권6호
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• pp.3631-3634
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• 2013

MicroRNAs (MiRNAs) play important roles in coordinating a variety of cellular processes and abnormal expression has been linked to the occurrence of several cancers. The miRNA miR-451 is downregulated in colorectal carcinoma (CRC) cells, suggested by several research groups including our own. In this study, synthetic miR-451 mimics were transfected into the SW620 human CRC cell line using Lipofectamine 2000 and expression of miR-451 was analyzed by real time PCR, while expression of CAB39, LKB1, AMPK, AKT, PI3K and Bcl2 was analyzed by Western blot, and cell growth was detected by MTT assay. In comparison to the controls, a significant increase in the expression of miR-451 was associated with significantly decreased expression of CAB39, LKB1, AMPK, AKT, PI3K and Bcl2. The capacity of cell proliferation was significantly decreased by miR-451 expression, which also inhibited cell growth. Our study confirmed that miR-451 has a repressive role in CRC cells by inhibiting cell growth through down-regulating the P13K/AKT pathway.

• 대한의생명과학회지
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• 제28권3호
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• pp.200-205
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• 2022

Established cancer cell lines are widely used for developing biomarkers for the patient-specific treatment of colorectal cancer and predicting prognoses. However, cancer cell lines may exhibit different drug responses depending upon the characteristics of the cell line. Therefore, it is necessary to select a tumor cell line suitable for the purpose of the study by considering the cell characteristics. This study investigated the levels of CXCL10, which were recently been reported to play an important role in the outcome of tumor treatment, secreted by colon cancer cells. 2 × 10⁵ cells/mL of each colorectal cancer cell was seeded into a 35 mm cell culture dish. After 24 h incubation, culture supernatant was used to determine the secreted CXCL10 levels. Among six colorectal cancer cell lines (HT-29, HCT116, CaCo-2, SW620, SW480, and CT26), Caco-2 cells showed the highest level of CXCL10 secretion. HT-29 cells showed the second-highest level of CXCL10 secretion. No significantly measurable level of CXCL10 secretion was detected in HCT116 cells. These results will be helpful in investigating the molecular basis of colorectal cancer.

• Asian Pacific Journal of Cancer Prevention
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• 제15권8호
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• pp.3767-3772
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• 2014

Introduction: MicroRNAs have emerged as post-transcriptional regulators that are critically involved in tumorigenesis. This study was designed to explore the effect of miRNA 133b on the proliferation and expression of TBPL1 in colon cancer cells. Methods: Human colon cancer SW-620 cells and human colon adenocarcinoma HT-29 cells were cultured. MiRNA 133b mimcs, miRNA 133b inhibitors, siRNA for TBPL1 and scrambled control were synthesized and transfected into cells. MiR-133b levels in cells and CRC tumor tissue was measured by real-time PCR. TBPL1 mRNA was detected by RT-PCR. Cell proliferation was studied with MTT assay. Western blotting was applied to detect TBPL1 protein levels. Luciferase assays were conducted using a pGL3-promoter vector cloned with full length of 3'UTR of human TBPL1 or 3'UTR with mutant sequence of miR-133b target site in order to confirm if the putative binding site is responsible for the negative regulation of TBPL1 by miR-133b. Results: Real time PCR results showed that miRNA 133b was lower in CRC tissue than that in adjacent tissue. After miR-133b transfection, its level was elevated till 48h, accompanied by lower proliferation in both SW-620 and HT-29 cells. According to that listed in http://www.targetscan.org, the 3'-UTR of TBPL1 mRNA (NM_004865) contains one putative binding site of miR-133b. This site was confirmed to be responsible for the negative regulation by miR-133b with luciferase assay. Further, Western blotting and immunohistochemistry both indicated a higher TBPL1 protein expression level in CRC tissue. Finally, a siRNA for TBPL1 transfection obviously slowed down the cell proliferation in both SW-620 and HT-29 cells. Conclusion: MiR-133b might act as a tumor suppressor and negatively regulate TBPL1 in CRC.

• Molecules and Cells
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• 제41권5호
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• pp.423-435
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• 2018

This investigation was aimed at working out the combined role of lncRNA H19, miR-29b and Wnt signaling in the development of colorectal cancer (CRC). In the aggregate, 185 CRC tissues and corresponding para-carcinoma tissues were gathered. The human CRC cell lines (i.e. HT29, HCT116, SW480 and SW620) and normal colorectal mucosa cell line (NCM460) were also purchased. Si-H19, si-NC, miR-29b-3p mimics, miR-29b-3p inhibitor, si-PGRN and negative control (NC) were, respectively, transfected into the CRC cells. Luciferase reporter plasmids were prepared to evaluate the transduction activity of $Wnt/{\beta}-catenin$ signaling pathway, and dual-luciferase reporter gene assay was arranged to confirm the targeted relationship between H19 and miR-29b-3p, as well as between miR-29b-3p and PGRN. Finally, the proliferative and invasive capacities of CRC cells were appraised through transwell, MTT and scratch assays. As a result, overexpressed H19 and down-expressed miR-29b-3p displayed close associations with the CRC patients' poor prognosis (P < 0.05). Besides, transfection with si-H19, miR-29b-3p mimic or si-PGRN were correlated with elevated E-cadherin expression, decreased snail and vimentin expressions, as well as less-motivated cell proliferation and cell metastasis (P < 0.05). Moreover, H19 was verified to directly target miR-29b-3p based on the luciferase reporter gene assay (P < 0.05), and miR-29b-3p also bound to PGRN in a direct manner (P < 0.05). Finally, addition of LiCl ($Wnt/{\beta}-catenin$ pathway activator) or XAV93920 ($Wnt/{\beta}-catenin$ pathway inhibitor) would cause remarkably altered E-cadherin, c-Myc, vimentin and snail expressions, as well as significantly changed transcriptional activity of ${\beta}-catenin/Tcf$ reporter plasmid (P < 0.05). In conclusion, the lncRNA H19/miR-29b-3p/PGRN/Wnt axis counted a great deal for seeking appropriate diagnostic biomarkers and treatment targets for CRC.