Synergistic inhibition of sunitinib and ethaselen against human colorectal cancer cells proliferation

Sunitinib, a multi-targeted tyrosine kinase inhibitor, has been widely used in the therapy of advanced renal cell cancer and imatinib-resistant gastrointestinal stromal tumors. However, little benefits could be obtained from sunitinib for patients with other types of solid tumors including colorectal cancer (CRC). Ethaselen (BBSKE), a specific thioredoxin reductase 1 inhibitor, has shown convincing anticancer effects both in vivo and in vitro. In this study, we explored the combinatory effect of sunitinib and BBSKE in human CRC cell lines LoVo, HT-29 and RKO. Cotreatment of BBSKE and sunitinib with the ratio of 2:1 for 24 h displayed synergistic effect against CRC cells proliferation. Apoptosis analysis also revealed that combination treatment of BBSKE and sunitinib (2:1) for 24 h induced higher apoptosis rate than either single treatment. The synergistic effect against LoVo cells proliferation may be explained by sharp reduction of Bcl-2/Bax protein expression ratio, decrease of pro-Caspase-3 protein expression along with significantly augmented Caspase-3 enzymatic activity, and release of cytochrome C from mitochondria to cytoplasm in the combination treatment group. The significant inhibition of vascular endothelial growth factor receptor 2 (VEGFR2) phosphorylation might also account for the synergism in cotreatment group. In short, sunitinib plus BBSKE is perhaps a promising strategy for colorectal cancer therapy.     

Long noncoding RNA LINC01296 plays an oncogenic role in colorectal cancer by suppressing p15 expression

ObjectiveTo examine the role of the long noncoding RNA LINC01296 in colorectal carcinoma (CRC) and to explore the underlying mechanism.MethodsWe detected LINC01296 expression levels in a cohort of 51 paired CRC and normal tissues. We also assessed the effects of LINC01296 on cell proliferation and apoptosis in CRC cells in vitro, and measured its effect on tumor growth in an in vivo mouse model. We identified the potential downstream targets of LINC01296 and assessed its regulatory effects.ResultsExpression levels of LINC01296 were elevated in 37/51 CRC tissues compared with the corresponding normal tissues and were significantly associated with tumor stage, lymph node metastasis, and distant metastasis. Knockdown of LINC01296 using antisense oligonucleotides inhibited cell proliferation and promoted apoptosis of colon cancer cells in vitro and inhibited tumor growth in vivo. Knockdown of LINC01296 also significantly increased the gene expression of p15 in colon cancer cells. LINC01296-specific suppression of p15 was validated by the interaction between enhancer of zeste homolog 2 and LINC01296.ConclusionOverexpression of LINC01296 suppressed the expression of p15 leading to CRC carcinogenesis. These findings may provide the basis for novel future CRC-targeted therapies.     

Long non coding RNA UCA1 contributes to the autophagy and survival of colorectal cancer cells via sponging miR-185-5p to up-regulate the WISP2/β-catenin pathway

The estimated number of new cases of colorectal cancer (CRC) will increase to 140 250 in 2018 worldwide. The long non-coding RNA (lncRNA) urothelial carcinoma-associated 1 (UCA1) has recently been shown to be dysregulated in CRC, which plays an important role in the progression of CRC. However, the biological role and the underling mechanism of UCA1 in the carcinogenesis of CRC remain unclear. Herein, we found that UCA1 was aberrantly upregulated in two CRC cell lines (SW620 and HT29) compared to colorectal cell CCD-18Co. UCA1 knockdown inhibited the apoptosis, growth and autophagy of CRC cell lines in vitro. Furthermore, UCA1 could act as an endogenous sponge by directly interacting with miR-185-5p and downregulation miR-185-5p expression. In addition, UCA1 could reverse the inhibitory effect of miR-185-5p on the growth and autophagy of CRC cells, which might be involved in the derepression of member 1 (WNT1)-inducible signaling pathway protein 2 (WISP2, a target gene of miR-185-5p) expression and the activation of the WISP2/β-catenin signaling pathway. In vivo, the present study elucidates a novel UCA1-miR-185-5p-WISP2-Wnt/β-catenin axis in CRC, which may help us to understand the pathogenesis and the feasibility of lncRNA-directed diagnosis and therapy of CRC.

The estimated number of new cases of colorectal cancer (CRC) will increase to 140 250 in 2018 worldwide.     

Pristimerin inhibits proliferation,migration and invasion,and induces apoptosis in HCT-116 colorectal cancer cells

Colorectal cancer (CRC) is one of the world’s most common cancers with a high mortality rate mainly due to metastasis. Our previous study showed that pristimerin had potent antitumor activities against human CRC cells. In the present study, we further evaluated pristimerin anti-tumor and anti-metastatic properties. MTT assay, Hoechst staining, Annexin V/PI double staining, reactive oxygen species (ROS) measurements were used to assess pristimerin cytotoxicity and apoptotic-inducing effects on HCT-116 cells. Wound healing assay and Transwell assay were used to estimate pristimerin anti-migration and anti-invasion activities on CRC cells. Meanwhile, HCT-116 xenograft model applied for investigating in vivo antitumor activities. Our results showed that pristimerin mediated in vitro HCT-116 cell death, through generation of intracellular ROS and apoptosis induction. Tumor volumes and weights measurements, pathological analysis and Tunnel assay proved that pristimerin inhibited in vivo HCT-116 xenografts growth. Pristimerin was also able to limit CRC invasion and metastasis. It caused downregulation of PI3K/AKT/mTOR pathway and its subsequent downstream p70S6K and E4-BP1 proteins. Collectively, pristimerin exerted both in vitro and in vivo cytotoxic and anti-metastatic effects on HCT-116 cells, suggesting that pristimerin has potential as a new anticancer drug for treatment of colon cancer.     

Retracted Article: Daphnetin inhibits proliferation and glycolysis in colorectal cancer cells by regulating the PI3K/Akt signaling pathway

Daphnetin (7,8-dihydroxycoumarin), a natural coumarin compound, has shown antitumor and energy metabolism regulatory activities. However, the effects of daphnetin on cell proliferation, migration, and glucose metabolism in colorectal cancer (CRC) cells remains unknown. In this study, the effects of daphnetin on CRC cell proliferation, migration, and glucose metabolism have been examined. The results showed that daphnetin inhibited the proliferation, migration, and invasion of CRC cells, and induced CRC cell apoptosis. Furthermore, daphnetin suppressed intracellular glucose and lactate production, and downregulated the expression of hexokinase 2 (HK2) and glucose transporter 1 (GLUT1) in CRC cells. Furthermore, daphnetin prevented activation of the PI3K/Akt pathway in CRC cells. These findings demonstrated that daphnetin inhibited the proliferation, migration and glucose metabolism in CRC cells by suppressing the PI3K/Akt signaling pathway. Therefore, daphnetin has potential as a novel anticancer agent for CRC treatment.

Daphnetin (7,8-dihydroxycoumarin), a natural coumarin compound, has shown antitumor and energy metabolism regulatory activities.     

蟾蜍灵在诱导HL-60细胞凋亡时对SYK和CBL家族蛋白的影响

为了探讨酪氨酸蛋白激酶SYK和CBL家族泛素连接酶在蟾蜍灵诱导HL-60细胞凋亡过程中的作用机制,采用台盼蓝染色检测细胞活力,采用流式细胞术检测细胞凋亡,采用Western blot和免疫沉降技术检测CBL、CBL—b表达和SYK的磷酸化。结果显示,蟾蜍灵以时间和剂量依赖方式抑制HL-60细胞增殖,24、48和72小时抑制细胞活力的IC50浓度分别约为26．3,7．8和2．0 nmol／L。高剂量蟾蜍灵从8小时即开始诱导HL-60细胞凋亡;与此同时蟾蜍灵快速活化SYK,并以时间依赖的方式下调CBL和CBL—b表达。结论：SYK活化及CBL家族蛋白表达下调可能参与蟾蜍灵对HL-60细胞的增殖抑制和凋亡诱导作用。     

蟾蜍灵对HL—60细胞的生长抑制及凋亡诱导作用

目的 研究中药蟾酥有效成分蟾蜍灵（ｂｕｆａｌｉｎ）对人白血病细胞的作用及其机制。方法 应用ＭＴＴ比色法观察蟾蜍灵对细胞的抑制作用。荧光显微境和透射电镜观察细胞结果色的改变,ＤＮＡ交电泳１、原位缺口标记法和流式细胞术等分析细胞凋亡。结果 ①蟾蜍灵明显抑制ＨＬ－６０细胞的生长,半数抑制浓度（ＩＣ５０）约为０．０２５μｍｏｌ／Ｌ;②典型的细胞形态学改变、ＤＮＡ片段化和亚Ｇ１峰的检出等证实蟾蜍灵能诱导白血     

KITENIN-targeting MicroRNA-124 Suppresses Colorectal Cancer Cell Motility and Tumorigenesis

MicroRNAs are increasingly implicated in the modulation of the progression of various cancers. We previously observed that KAI1 C-terminal interacting tetraspanin (KITENIN) is highly expressed in sporadic human colorectal cancer (CRC) tissues and hence the functional KITENIN complex acts to promote progression of CRC. However, it remains unknown that microRNAs target KITENIN and whether KITENIN-targeting microRNAs modulate CRC cell motility and colorectal tumorigenesis. Here, through bioinformatic analyses and functional studies, we showed that miR-124, miR-27a, and miR-30b negatively regulate KITENIN expression and suppress the migration and invasion of several CRC cell lines via modulation of KITENIN expression. Through in vitro and in vivo induction of mature microRNAs using a tetracycline-inducible system, miR-124 was found to effectively inhibit the invasion of CT-26 colon adenocarcinoma cells and tumor growth in a syngeneic mouse xenograft model. Constitutive overexpression of precursor miR-124 in CT-26 cells suppressed in vivo tumorigenicity and resulted in decreased expression of KITENIN as well as that of MYH9 and SOX9, which are targets of miR-124. Thus, our findings identify that KITENIN-targeting miR-124, miR-27a, and miR-30b function as endogenous inhibitors of CRC cell motility and demonstrate that miR-124 among KITENIN-targeting microRNAs plays a suppressor role in colorectal tumorigenesis.     

Effect of resveratrol on drug resistance in colon cancer chemotherapy

To investigate the effects of resveratrol on the drug resistance of 5-FU in the colon cancer chemotherapy, an MTT assay was used to detect the effects of 5-FU and resveratrol combined with 5-FU on the proliferation of the LoVo and SW480 cell lines. Flow cytometry was used to detect the effect of 5-FU combined with resveratrol on the survival rate of the LoVo and SW480 cells. A western blot was used to detect the expression levels of the proteins associated with colon cancer. After flow sorting, the percentage of the SW480 and the LoVo cell line CD133⁺ was 97.5% and 95.8%, respectively. The cells cultured in vitro showed more rapid cell proliferation and differentiation. The MTT assay showed that as compared with the survival rate of the blank group LoVo and CD133⁺ LoVo cells, the survival rate of the cells containing the 5-FU group was lower (P < 0.05). When 5-FU was used in combination with different concentrations of resveratrol, the abovementioned phenomenon was more prominent. The sorted colon cancer cells have dry stem cells, and the sorted CD133⁺ cells are more resistant to drugs; the combination of resveratrol and 5-FU has the best effect on the colon cancer cells. Preliminary studies on the mechanism of action of the drug show that a combination of 5-FU and resveratrol regulates apoptosis in CD133⁺ colon cancer stem cells by regulating the BAX gene; however, more complex mechanisms may also be involved.

To investigate the effects of resveratrol on the drug resistance of 5-FU in the colon cancer chemotherapy, an MTT assay was used to detect the effects of 5-FU and resveratrol combined with 5-FU on the proliferation of the LoVo and SW480 cell lines.     

蟾蜍灵在诱导HL-60细胞凋亡过程中对Bcl-2和PKC的影响

为了研究蟾蜍灵在诱导HL-60细胞凋亡过程中对Bcl-2表达与裂解和磷酸化的作用,及对蛋白激酶C(PKC)活性与PKCs亚细胞定位的影响,分别采用台盼蓝拒染法检测细胞生存率,细胞染色法观察凋亡形态,流式细胞术分析细胞周期,琼脂糖凝胶电泳检测凋亡DNA片段化,[γ-32P]同位素掺入法测定PKC活性,Westernblot分析Bcl-2及PKC蛋白表达。结果表明:①蟾蜍灵抑制HL-60细胞增殖,24、48及72小时的IC50分别为(25.8±2·1)、(8.0±1.2)及(2.3±0.3)nmol/L;②50nmol/L蟾蜍灵作用24小时可诱导HL-60细胞凋亡;③50nmol/L蟾蜍灵处理HL-60细胞6-24小时,Bcl-2蛋白表达水平明显下调,并出现23kD的裂解片段,磷酸化水平逐渐降低;④1-100nmol/L蟾蜍灵分别作用30分钟,对PKC总活性无影响,但可促使PKCβⅡ膜转位。结论:蟾蜍灵诱导HL-60细胞凋亡可能与Bcl-2表达降低、裂解及脱磷酸化有关。     

miR-142-3p靶向Wnt/β-链蛋白通路对结直肠癌细胞增殖的影响

目的探究miR-142-3p靶向Wnt/β-链蛋白(β-catenin)通路对结直肠癌(CRC)细胞增殖的影响。方法选取2018年1月—2020年10月收治的66例CRC的肿瘤组织及其相邻正常组织。同时培养正常人结肠上皮细胞NCM460和CRC细胞系(HT29、LoVo、HCT116、Caco2、SW480细胞)。通过qRT-PCR检测CRC肿瘤组织、正常组织、正常人结肠上皮细胞与CRC细胞系中miR-142-3p表达水平;经免疫蛋白印迹法检测CRC细胞系中β-catenin表达水平;采用CCK-8法和流式细胞术检测miR-142-3p过表达后对CRC细胞增殖的影响;经免疫蛋白印迹检测过表达miR-142-3p对Wnt/β-catenin信号通路相关蛋白的影响;采用双荧光素酶报告基因检验miR-142-3p与β-catenin编码基因CTNNB1的靶向关系。结果miR-142-3p在CRC肿瘤组织和CRC细胞系中的表达显著下降(P<0.05);β-catenin在CRC细胞系中的表达显著升高(P<0.05);过表达miR-142-3p可靶向结合CTNNB1,显著抑制Caco2、LoVo和HT29细胞的增殖和Ki67+细胞比例,抑制β-catenin、c-myc和Cyclin D1的表达(P<0.05)。结论miR-142-3p可通过靶向调节β-catenin的表达,干扰Wnt/β-catenin通路,抑制CRC细胞的增殖。     

Bufalin,a bufanolide steroid from the parotoid glands of the Chinese toad,inhibits L‐type Ca2+ channels and contractility in rat ventricular myocytes

Bufalin is a bufanolide steroid compound in Chan Su. Chan Su is a traditional Chinese medicine prepared from the dried white secretion of the auricular and skin glands of toads and has been used as an oriental drug. However, the effect of bufalin on cardiac function and its underlying cellular mechanisms remain unclear. Here, we explore the cellular mechanisms of bufalin on myocardial protection via the whole‐cell patch‐clamp recording and video‐based edge detection system. Exposure to bufalin resulted in a concentration‐dependent blockade of I_Ca‐L, with the half‐maximal inhibitory concentration (IC₅₀) of 60 μm and the maximal inhibitory effect of 71.50 ± 2.67%. Bufalin at 100 μm reduced cell shortening by 33.83 ± 4.01%. Bufalin restrained L‐type Ca²⁺ channels conductance, and contractility in rat ventricular myocytes. Thus, the protective effects of bufalin on the heart may be determined by the inhibitory effect on I_Ca‐L and the negative inotropic action caused by the decrease of intracellular Ca²⁺ in rat myocardial cells.     

Coptisine suppresses tumor growth and progression by down-regulating MFG-E8 in colorectal cancer

Treating colorectal cancer (CRC) continues to be a clinical challenge. Coptisine, an alkaloid derived from Coptis chinensis Franch. shows toxic effects on CRC cells, but its underlying mechanism remains elusive. MFG-E8 is involved in tumor growth and progression. Herein, we evaluated the effects of coptisine on MFG-E8 in CRC, and explored the mechanism. The expression of MFG-E8 in CRC and adjacent normal colon tissue samples from patients was detected. The effects of coptisine on CRC cells HCT116 in vitro were evaluated by CCK-8, adhesion and transwell assays. A xenograft tumor model was used to assess the effects of coptisine in vivo. The morphology of CRC tissue was observed by HE staining. Cell signaling was tested using western blotting and immunohistochemical assay. The expression of MFG-E8 in human CRC tissue samples significantly increased compared with that of adjacent normal ones. Coptisine significantly reduced the expressions of MFG-E8 in HCT116 cells and tumor-bearing mice. Moreover, coptisine suppressed the growth, adhesion and metastasis of CRC cells. Coptisine also suppressed the expression of MMP-2 and MMP-9 via the PI3K/AKT signaling pathway. Furthermore, it inhibited epithelial–mesenchymal transition in vivo and in vitro. Coptisine inhibited CRC growth and progression by down-regulating MFG-E8, and is a potential candidate for treatment.

Treating colorectal cancer (CRC) continues to be a clinical challenge. Coptisine, an alkaloid derived from Coptis chinensis Franch. shows toxic effects on CRC cells, but its underlying mechanism remains elusive.     

早幼粒细胞白血病蛋白与结直肠腺瘤癌变的相关性及机制初探

目的 分析结直肠腺瘤、结直肠癌组织及肠癌细胞中早幼粒细胞白血病蛋白(promyelocytic leukemia protein,PML)的表达情况,探讨PML在结直肠癌发生发展中的作用.方法 采用免疫组化、Western印迹法检测PML在结直肠癌、结直肠腺瘤及正常结直肠组织中的表达情况.检测10种不同肠癌细胞株的PM...     

蟾蜍灵诱导K562细胞分化和凋亡过程中WT1表达的下调

目的：研究蟾蜍灵在诱导K562细胞分化和凋亡中对WT1基因的凋节作用。方法：采用锥虫蓝拒染法测定细胞增殖活力；采用形态学观察、流式细胞术分析和NBT还原试验检测细胞分化和细胞凋亡；采用Western blot和RT－PCR检测WT1蛋白和mRNA的表达。结果：（1）蟾蜍灵抑制K562细胞的增殖，24，48和72h的IC50分别为0．026，0．032和0．006μmol/L。（2）蟾蜍灵浓度在0．01－0．05μmol/L可明显诱导K562细胞向单核／巨噬细胞分化，大于0．260μmol/L 时可明显诱导细胞凋亡。（3）细胞分化早期和细胞凋亡发生前，蟾蜍灵明显下调K562细胞WT1 mRNA和蛋白的表达。结论：蟾蜍灵诱导K562细胞向单核／巨噬细胞分化和细胞凋亡可能与其对WT1的下调有关。     

Recent developments in targeting genes and pathways by RNAi‐based approaches in colorectal cancer

A wide spectrum of genetic and epigenetic variations together with environmental factors has made colorectal cancer (CRC), which involves the colon and rectum, a challenging and heterogeneous cancer. CRC cannot be effectively overcomed by common conventional therapies including surgery, chemotherapy, targeted therapy, and hormone replacement which highlights the need for a rational design of novel anticancer therapy. Accumulating evidence indicates that RNA interference (RNAi) could be an important avenue to generate great therapeutic efficacy for CRC by targeting genes that are responsible for the viability, cell cycle, proliferation, apoptosis, differentiation, metastasis, and invasion of CRC cells. In this review, we underline the documented benefits of small interfering RNAs and short hairpin RNAs to target genes and signaling pathways related to CRC tumorigenesis. We address the synergistic effects of RNAi‐mediated gene knockdown and inhibitors/chemotherapy agents to increase the sensitivity of CRC cells to common therapies. Finally, this review points new delivery systems/materials for improving the cellular uptake efficiency and reducing off‐target effects of RNAi.     

异甘草素对结直肠癌细胞的体内外抑制作用研究

目的探讨异甘草素(ISL)对结直肠癌细胞的体外增殖、细胞凋亡、细胞周期进程和细胞迁移能力的影响,以及对小鼠结直肠癌移植瘤生长的抑制作用。方法采用溴化噻唑蓝四氮唑(MTT)法、流式细胞术、划痕实验分别检测ISL作用后,结直肠癌HCT-116细胞及SW480细胞的增殖率、凋亡率、细胞周期分布及细胞迁移的变化。复制小鼠结直肠癌移植瘤模型,观察肿瘤生长,同时通过HE染色分析ISL干预移植瘤后的病理改变情况。结果ISL干预后HCT-116细胞及SW480细胞增殖率明显下降(P<0.01),G0/G1期细胞的比例降低(P<0.01),S期细胞的比例升高(P<0.05),细胞凋亡增多(P<0.05),同时细胞划痕的间隙增大,细胞迁移的距离较短。经ISL处理后,小鼠移植瘤的生长也受到显著抑制(P<0.05)。结论ISL可促使结直肠癌细胞发生凋亡,细胞周期于S期阻滞及细胞迁移力下降,同时可抑制结直肠癌细胞的体外增殖及体内移植瘤生长。     

Odontogenic ameloblast-associated protein (ODAM) inhibits human colorectal cancer growth by promoting PTEN elevation and inactivating PI3K/AKT signaling

Odontogenic ameloblast-associated protein (ODAM), an acidic matricellular protein, has been implicated in several epithelial neoplasms. However, its biological functions and molecular mechanisms in cancer progression, particular colorectal carcinoma (CRC), remain unknown. Here we demonstrated that ODAM was significantly down-regulated in CRC tissues compared with their normal counterparts. Then, we established that ODAM expression level was closely correlated with CRC development and patient prognosis. The abnormal expression of ODAM dramatically affected CRC cell growth in vitro and in vivo. We further revealed that the inhibitory effects of ODAM on CRC cell growth were associated with PTEN elevation and PI3K/AKT signaling inactivation. Furthermore, we determined that silencing of PTEN expression yielded recovery of AKT activity in ODAM-expressing CRC cells. Our study suggests matricellular protein ODAM may serve as a novel prognostic marker and act as a CRC growth suppressor.     

Effect of CLN3 silencing by RNA interference on the proliferation and apoptosis of human colorectal cancer cells

Apoptosis constitutes a system for the removal of aged, or damaged cells, which is regulated by the interplay of pro-apoptotic and antiapoptotic proteins. Previous study has shown that Juvenile Batten disease protein, CLN3, is antiapoptotic gene in NT2 neuronal precursor cells and a few types of cancers. However, in colorectal cancer, whether CLN3 also play its antiapoptotic role and the effect of targeted controlling CLN3 on the biological behavior of human colorectal cancer cell is unknown. We employed the sequence-specific siRNA silencing the CLN3 gene and investigated its effects on growth and apoptosis of colorectal cancer HCT116 cells, which has highest elevation of CLN3 expression among four colorectal cancer cell lines. After CLN3 specific siRNA transfection, mRNA and protein expression levels of CLN3 in HCT116 cells were noticeably decreased. Moreover, CLN3-siRNA inhibited the proliferation of colorectal cancer cells, promoted their apoptosis and induced G0/G1 cell cycle arrest. Our current study demonstrated that CLN3 was expressed in colorectal cancer cells at a high frequency. Moreover, CLN3 down-regulation with RNA interference can inhibit proliferation, apoptosis, and cell cycle progression of colorectal cancer cells. Our study represented a potential new approach to understanding the role of CLN3 in cancer and provides a potential novel strategy colorectal cancer therapy.