Inhibition of acrolein-stimulated MUC5AC expression by Platycodon grandiflorum root-derived saponin in A549 cells

Mucin overproduction is a hallmark of chronic airway diseases such as chronic obstructive pulmonary disease. In this study, we investigated the inhibition of acrolein-induced expression of mucin 5, subtypes A and C (MUC5AC) by Changkil saponin (CKS) in A549 cells. Acrolein, a known toxin in tobacco smoke and an endogenous mediator of oxidative stress, increases the expression of airway MUC5AC, a major component of airway mucus. CKS, a Platycodon grandiflorum root-derived saponin, inhibited acrolein-induced MUC5AC expression and activity, through the suppression of NF-κB activation. CKS also repressed acrolein-induced phosphorylation of ERK1/2, JNK1/2, and p38MAPK, which are upstream signaling molecules that control MUC5AC expression. In addition, the MAPK inhibitors PD98059 (ERK1/2), SP600125 (JNK1/2), and SB203580 (p38 MAPK), and a PKC delta inhibitor (rottlerin; PKCδ) inhibited acrolein-induced MUC5AC expression and activity. CKS repressed acrolein-induced phosphorylation of PKCδ. Moreover, a reactive oxygen species (ROS) inhibitor, N-acetylcysteine, inhibited acrolein-induced MUC5AC expression and activity through the suppression of PKCδ and MAPK activation, and CKS repressed acrolein-induced ROS production. These results suggest that CKS suppresses acrolein-induced MUC5AC expression by inhibiting the activation of NF-κB via ROS-PKCδ-MAPK signaling.     

Mechanisms of thiosulfinates from Allium tuberosum L.-induced apoptosis in HT-29 human colon cancer cells

This study was performed to elucidate the apoptotic pathways by thiosulfinates, major biologically active components of Allium tuberosum L., in HT-29 human colon cancer cells. Thiosulfinates significantly induced cell death in dose- and time-dependent manners in HT-29 cells, which is associated with apoptosis. Thiosulfinates activated the initiator caspase-8, and -9, and the effector caspase-3. In the present study, thiosulfinates were found to stimulate Bid cleavage, indicating that the apoptotic action of caspase-8-mediated Bid cleavage leads to the activation of caspase-9. Thiosulfinates down-regulated the expression of the anti-apoptotic protein Bcl-2, and up-regulated the expression of the pro-apoptotic protein Bax. We also found that thiosulfinates increased the expression of AIF, a caspase-independent mitochondrial apoptosis factor, and induced DNA fragmentation and chromatin condensation in HT-29 cells. These results indicate that thiosulfinates from A. tuberosum L. inhibited cell proliferation and activated both the caspase-dependent and caspase-independent apoptotic pathways in HT-29 cells.     

活性氧介导5, 7- 二甲氧基黄酮诱导结肠癌HT- 29 细胞凋亡

目的研究5,7-二甲氧基黄酮（5,7-dimethoxyflavone,DMF）诱导体外培养人结肠癌HT-29细胞凋亡作用及其机制。方法以HT-29细胞为研究对象,利用ELISA法测定细胞DNA断裂,流式细胞术检测细胞凋亡率和活性氧（ROS）水平。结果DMF激活HT-29细胞ROS生成（P〈0．05）,呈浓度依赖性。DMF以浓度依赖方式促进HT-29细胞DNA断裂（P〈0．05）,同时,诱导细胞凋亡率增高（P〈0．05）;10μmol．L^-1N-乙酰半胱氨酸预孵育30min,能有效阻断DMF诱导ROS生成和细胞凋亡作用（P〈0．05）。结论DFM通过促进结肠癌HT-29细胞ROS生成诱导细胞凋亡。     

雀梅藤石油醚提取物诱导三阴性乳腺癌细胞凋亡的实验研究

目的 探讨雀梅藤石油醚提取物对乳腺癌细胞增殖和凋亡的影响。方法 石油醚提取物孵育乳腺癌细胞不同时间后,CCK8法检测细胞活力,平板克隆形成法检测细胞增殖,DAPI染色法观察细胞核形态变化,免疫荧光检测线粒体膜电位和活性氧变化,流式细胞术检测细胞周期和凋亡,CCK8法观察对人正常血管内皮细胞毒性。结果 雀梅藤石油醚提取物孵育BT549和MDA-MB-231人三阴性乳腺癌细胞株24 h后的IC₅₀分别为45.40 μg/ml和12.23 μg/ml,时间和剂量依赖地抑制乳腺癌细胞活力和克隆形成,诱导细胞凋亡,阻滞细胞周期于G1/S期,降低线粒体膜电位,升高活性氧;对人正常血管内皮细胞无明显毒性作用。结论 雀梅藤石油醚提取物可能通过升高活性氧,引起线粒体膜电位坍塌,激活线粒体通路诱导细胞凋亡,从而抑制乳腺癌细胞生长,支持其民间抗乳腺肿瘤的应用。     

Anti-proliferative effects of Lethariella zahlbruckneri extracts in human HT-29 human colon cancer cells

This study was performed to elucidate the anti-proliferative effects and the apoptotic mechanisms of extracts from Lethariella zahlbruckneri in HT-29 human colon cancer cells. Both the acetone extract (AEL) and methanolic extract (MEL) of L. zahlbruckneri decreased viable cell numbers in a dose- and time-dependent manner in HT-29 cells. The AEL showed stronger cytotoxicity than MEL. Cell death induced by AEL increased cell populations in the sub-G1 phase, as well as the formation of apoptotic bodies and nuclear condensation, whereas MEL did not. Therefore, the potential of AEL to induce apoptosis was examined. Apoptosis induced by AEL was associated with the activation of initiator caspases-8 and -9, as well as the effector caspase-3. AEL stimulated Bid cleavage. This indicated that the apoptotic action of caspase-8-mediated Bid cleavage leads to the activation of caspase-9. AEL increased the expression of the pro-apoptotic protein, Bax, and decreased the expression of the anti-apoptotic protein, Bcl-2. AEL also increased the expression of the caspase-independent mitochondrial apoptosis factor, AIF, in HT-29 cells. These results indicate that AEL inhibited HT-29 cell proliferation by inducing apoptosis, which might be mediated via both caspase-dependent and -independent pathways.     

Glossogin, a novel phenylpropanoid from Glossogyne tenuifolia, induced apoptosis in A549 lung cancer cells

Glossogyne tenuifolia has been shown to exhibit good antioxidant and anticancer activity. In this study, a new phenylpropanoid compound, glossogin (1′-acetoxy-4-O-isovalyryleugenol), was isolated from ethyl acetate extract of G. tenuifolia by using column chromatography and HPLC. Its chemical structure was determined by ¹H and ¹³C NMR, MS and IR spectroscopic evidence. This compound showed the cytotoxicity against A549 human lung cancer cell line and it induced the progressing apoptosis on A549 cells. This apoptosis was verified as A549 cells were arrested at the sub-G₁ phase. The apoptosis was accompanied by release of cytochrome C and activation of caspase-9 and -3. It was also associated with the decrease in Bcl-2 and Bcl-xL protein levels, and the increase in Bad protein expression. Data analysis suggests glossogin exerted significant apoptotic effect on A549 cells through the mitochondrial pathway. Hence, our findings showed that glossogin exhibited potential anticancer activity against lung cancer through proliferating inhibition and apoptosis induction of cancer cells.     

Induction of apoptosis by Panduratin A isolated from Kaempferia pandurata in human colon cancer HT-29 cells

We previously showed that panduratin A isolated from an extract of Kaempferia pandurata (Zingiberaceae) was a strong inhibitor of cyclooxygenase-2 (COX-2) in RAW264.7 cells, suggesting a potential use of panduratin A as an anti-inflammatory agent. In the present study, we have investigated the effects of panduratin A on cytoplasmic levels of COX-2, as well as proliferation and apoptosis in human colon cancer cells HT-29. Cell proliferation and induction of apoptosis was determined by the MTT assay, DNA fragmentation measurement, flow cytometric analysis, nuclear staining and Western blotting. The MTT assay indicated that panduratin A exhibited cytotoxicity with an IC50 value of 28 microM. The cytotoxic effects of panduratin A were found to be accompanied by the dose-dependent induction of apoptosis as assessed by DNA fragmentation and apoptotic bodies. In addition, treatment with an apoptosis-inducing concentration of panduratin A resulted in cleavage of poly(ADP-ribose) polymerase (PARP) with a concomitant decrease in procaspase-3 protein. Our study provides evidence for cell growth inhibition and induction of apoptosis by panduratin A in human colon cancer cells, suggesting its potential use as a cancer chemopreventive and therapeutic agent.     

New dammarane-type saponin from roots of Panax notoginseng

A new ginsenoside and 22 known compounds were isolated from the roots of Panax notoginseng (Araliaceae). The structure of the new compound was elucidated from 2D-NMR and other spectral evidence.     

NO-donating aspirin inhibits both the expression and catalytic activity of inducible nitric oxide synthase in HT-29 human colon cancer cells

Nitric oxide-releasing aspirin (NO-ASA) is emerging as a potentially important chemopreventive agent against colon cancer. We examined in HT-29 human colon adenocarcinoma cells the effect of NO-ASA on the inducible form of nitric oxide synthase (NOS2), an enzyme implicated in colon carcinogenesis. NO-ASA inhibited in a time- and concentration-dependent manner the expression of NOS2 up to 70% compared to control (IC50 for this effect = 46 microM). NO-ASA also decreased the corresponding steady-state mRNA levels and this reduction preceded the reduction of protein levels by at least 6 h. NO-ASA also reduced the enzymatic activity of NOS2, as determined by a direct enzyme assay (maximal reduction = 80%) and by determining the accumulation of NO in the culture medium (IC50 for this effect = 36 microM). These effects of NO-ASA on NOS2 were paralleled by inhibition in cell growth (IC50 = 8.5 microM). These findings indicate that NO-ASA profoundly inhibits both the expression and enzymatic activity of NOS2 and suggest that these effects may represent an important mechanism for the colon cancer chemopreventive effect of NO-ASA.     

p-JNK在结肠癌组织中的表达及抑制JNK通路后HT-29细胞增殖和凋亡变化

目的研究p-JNK在结肠癌组织中的表达以及抑制JNK信号通路后人结肠癌HT-29细胞增殖和凋亡的变化。方法使用免疫组织化学方法检测50例结肠癌组织标本和50例正常结肠组织标本中p-JNK的表达情况并和临床资料进行比较分析;在人结肠癌细胞株HT-29中使用SP600125抑制JNK信号通路后,MTT法检测细胞增殖,TUNEL法检测凋亡。结果 p-JNK在结肠癌组织中的表达水平明显高于正常对照组织(P<0.05);并和淋巴结转移、肿瘤分化程度、肿瘤的侵犯深度相关(P<0.05);而与肿瘤部位无关。抑制JNK信号通路后,HT-29细胞中的p-JNK表达降低(P<0.05);增殖减少,凋亡增加(P<0.05)。结论在结肠癌组织中存在p-JNK的高表达,抑制JNK信号通路能降低人结肠癌细胞株HT-29细胞的增殖,并促进凋亡;JNK信号通路和结肠癌的发生发展有关。     

A glycoprotein from Laminaria japonica induces apoptosis in HT-29 colon cancer cells

We isolated a novel glycoprotein from the brown alga Laminaria japonica that has antiproliferative effects on HT-29 colon cancer cells. We also identified the mechanism by which this glycoprotein, named LJGP, induces apoptosis. MTS assays showed that LJGP inhibited the proliferation of several cancer cell lines (AGS, HepG2, HT-29) in a dose-dependent manner. Especially in HT-29 cells, proliferation was significantly decreased. LJGP treatment on HT-29 displayed several apoptotic features, such as DNA fragmentation, sub-G1 arrest, caspase-3 activation, and PARP degradation. Consistent with sub-G1 arrest, LJGP decreased the expression of Cdk2, cyclin E, cyclin D1, PCNA, E2F-1, and phosphorylated pRb. Furthermore, the increase of p27 expression was observed. We also determined that LJGP-induced apoptosis leads to the formation of a death-induced signaling complex of Fas, FADD, and procaspase-8. LJGP induced the reduction of mitochondrial membrane potential with activation of the Bcl-2 family of proteins and caspase-9. These findings suggest that LJGP inhibits HT-29 cell proliferation by inducing apoptosis, which may be mediated via multiple pathways, including the Fas signaling pathway, the mitochondrial pathway, and cell cycle arrest. Therefore, LJGP can be a useful treatment option for colon cancer in humans.     

Structure-function relationship of the saponins from the roots of Platycodon grandiflorum for hemolytic and adjuvant activity

To assess the contribution of the aglycone and sugar chain to the biological activity of saponins from Platycodon grandiflorum, seven structurally consecutive saponins, platycodin D (PD), D2 (PD2), D3 (PD3), platycoside A (PA), E (PE), deapioplatycoside E (DPE), and polygalacin D2 (PGD) were compared for their hemolytic activities and adjuvant potentials on the immune responses to Newcastle disease virus-based recombinant avian influenza vaccine (rL-H5) in mice. Among seven compounds, the order of the hemolytic activity was PGD ≈ PD > PD2 > PA > PD3 > PE > DPE. PD, PD2, PA, and PGD significantly not only promoted concanavalin A (Con A)-, lipopolysaccharide (LPS)- and antigen-induced splenocyte proliferation, but enhanced the NK cell activity in mice immunized with rL-H5. PD and PD2 increased the antigen specific IgG, IgG1, IgG2a, and IgG2b antibody titers, while PA and PGD only induce the IgG and IgG1 antibody responses in the immunized mice. However, the other three saponins were not observed for adjuvant activity. The results suggested that the sugar chains attached to C-3, the glycidic moiety at C-28 of aglycone, as well as aglycone affect their biological activities. Interestingly, their hemolytic and adjuvant activities increased with the retention time by reverse phase HPLC analysis. The retention time may be useful for primary estimation of fundamental adjuvanticity of saponin with the same aglycone.     

Chemopreventive effect of protein extract ofAsterina pectinifera in HT-29 human colon adenocarcinoma cells

We investigated the effect of protein extract of Asterina pectinifera on the activity of 4 enzymes that may play a role in adenocarcinoma of the colon: quinone reductase (QR), glutathione S-transferase (GST), ornithine decarboxylase (ODC), and cyclooxygenase (COX)-2. QR and GST activity increased in HT-29 human colon adenocarcinoma cells increased that had been exposed to 4 concentrations of the protein extract (80, 160, 200, and 240 microg/mL). Additionally, 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ODC activity decreased significantly in cells exposed to the extract in concentrations of 160 microg/mL (p<0.05), 200 microg/mL (p<0.005), and 240 microg/mL (p<0.005). TPA-induced COX-2 activity also decreased in cells exposed to extract concentrations of 10, 20, 40, and 60 microg/mL. COX-2 expression was also inhibited in cells exposed to this extract. These results suggest that this protein extract of A pectinifera has chemopreventive activity in HT-29 human colon adenocarcinoma cells, and therefore, may have the potential to function as a chemopreventive agent in human colorectal cancer.     

Activated kRas protects colon cancer cells from cucurbitacin-induced apoptosis: the role of p53 and p21

Cucurbitacins have been shown to inhibit proliferation in a variety of cancer cell lines. The aim of this study was to determine their biological activity in colon cancer cell lines that do not harbor activated STAT3, the key target of cucurbitacin. In order to establish the role of activated kRas in the responsiveness of cells to cucurbitacins, we performed experiments in isogenic colon cancer cell lines, HCT116 and Hke-3, which differ only by the presence of an activated kRas allele. We compared the activity of 23, 24-dihydrocucurbitacin B (DHCB) and cucurbitacin R (CCR), two cucurbitacins that we recently isolated, with cucurbitacin I (CCI), a cucurbitacin with established antitumorigenic activity. We showed that cucurbitacins induced dramatic changes in the cytoskeleton (collapse of actin and bundling of tubulin microfilaments), inhibited proliferation and finally induced apoptosis of both HCT116 and Hke-3 cells. However, the presence of oncogenic kRas significantly decreased the sensitivity of cells to the three cucurbitacins tested, CCR, DHCB and CCI. We confirmed that mutational activation of kRas protects cells from cucurbitacin-induced apoptosis using nontransformed intestinal epithelial cells with inducible expression of kRasV12. Cucurbitacins induced the expression of p53 and p21 predominantly in HCT116 cells that harbor mutant Ras. Using HCT116 cells with targeted deletion of p53 or p21 we confirmed that p53 and p21 protect cells from apoptosis induced by cucurbitacins. These results demonstrated that sensitivity of human colon cancer cell lines to cucurbitacins depends on the kRas and p53/p21 status, and established that cucurbitacins can exert antitumorigenic activity in the absence of activated STAT3.     

Studies on the chemical constituents from the roots of <Emphasis Type="Italic">Platycodon grandiflorum</Emphasis>

Three known monodesmosidic saponins: 3-O--d-glucopyranosyl-2,3,16,23,24-pentahydroxyolean-12-ene-28-oic acid, 3-O--d-glucopyranosyl polygalacic acid, and 3-O--d-glucopyranosyl-(13)--d-glucopyranosyl polygalacic acid; and two known nonsaponin compounds: a mixed compound of n-tetracosanoic acid (lignoceric acid), n-hexacosanoic acid (cerotic acid), and n-octacosanoic acid, and -monopalmitin; were isolated for the first time from the root of Platycodon grandiflorum A. DC. together with another seven known compounds: platycoside G₁ (3-O--d-glucopyranosyl-(16)--d-glucopyranosyl-(16)--d-glucopyranosyl-2,3,16,23,24-pentahydroxyolean-12-ene-28-oic acid 28-O--d-xylopyranosyl-(14)--l-rhamnopyranosyl-(12)--l-arabinopyranoside), deapio-platycodin D, Polygalacin D, deapio-platycodin D₃, platycoside A, -spinasterol, and -spinasteryl-3-O--d-glucopyranoside. Alkaline hydrolysis of platycoside G₁ afforded a new monodesmosidic prosaponin: 3-O--d-glucopyranosyl-(16)--d-glucopyranosyl-(16)--d-glucopyranosyl-2,3,16,23,24-pentahydroxyolean-12-ene-28-oic acid. Their chemical structures were elucidated on the basis of their spectral data and chemical evidence.     

异叶败酱总苷片对人大肠癌HT-29细胞凋亡的影响

目的：研究异叶败酱总苷片对人大肠癌细胞凋亡的影响及作用机制。方法：建立人大肠癌HT-29裸鼠移植瘤模型，设立异叶败酱总苷片低、中、高剂量组和空白组、5-氟脲嘧啶（5-FU）＋甲酰四氢叶酸钙（CF）对照组，给药8周后处死裸鼠，瘤体称重,末端原位标记染色法（TUEL）检测凋亡指数，分别行半胱天冬氨酸蛋白酶-3（caspase-3），Bax和Bcl-2免疫组化染色，图象分析系统定量检测并比较该3种物质表达情况。结果：异叶败酱总苷片各组瘤重小于空白对照组，而凋亡指数均高于空白对照组和5-FU＋CF组，其中高剂量组与空白对照组和5-FU＋CF组比较差异有极显著性（P＜0．01）。异叶败酱总苷片各组移植瘤caspase-3表达及Bax、Bcl-2表达的相对比率（Bax／Bcl-2）均高于空白对照组。结论：异叶败酱总苷片具有诱导人大肠癌HT-29裸鼠移植瘤细胞凋亡的作用，其机制可能与促进移植瘤caspase-3表达及增大Bax／Bcl-2表达比率有关。     

槲寄生凝集素对人结肠癌HT-29细胞增殖及凋亡的影响

目的：研究槲寄生凝集素对人结肠癌HT-29细胞增殖和凋亡的影响。方法：采用MTT法观察槲寄生凝集素对体外培养的人结肠癌HT-29细胞增殖的影响,运用基因组DNA电泳观察凋亡特征性“梯状”条带及原位末端标记法检测槲寄生凝集素诱导HT-29细胞凋亡。结果：槲寄生凝集素对体外培养的人结肠癌HT-29细胞抑制作用表现出时间和剂量依赖性。基因组DNA琼脂糖凝胶电泳显示槲寄生凝集素作用于HT-29细胞后出现凋亡细胞特有的DNA阶梯状条带。原位末端标记法检测显示槲寄生凝集素可明显诱导HT-29细胞的凋亡。结论：槲寄生凝集素可抑制人结肠癌HT-29细胞生长并诱导其凋亡。     

Evaluation of isoindole derivatives: Antioxidant potential and cytotoxicity in the HT-29 colon cancer cells

Norcantharimides have an isoindole skeleton structure, and some isoindoline derivatives have positive effects on inflammatory pathologies, including cancers. The present study aims to evaluate the antioxidant and cytotoxic potential of four synthesized isoindoline derivatives (NCTD1–4). HT-29 cells exposed to 10, 50, 100, and 200 µM doses of each derivative were incubated for 24 and 48 h, respectively. The cytotoxicity of the new derivatives was analyzed using the cell growth inhibition assay and the cell membrane damage test. In vitro antioxidant activity studies showed that the derivatives have free radical-scavenging effects in a dose-dependent manner. NCTD3 and NCTD4 apparently have antioxidant effects when compared with the control group treated with dimethyl sulfoxide. Furthermore, NCTD4 inhibited the growth of the HT-29 cells due to membrane damage and exhibited a dose-dependent cytotoxic effect on colon adenocarcinoma cells. The findings suggest that NDTD4 has the highest potential for colon cancer treatment and may be interpreted as a candidate anticancer agent.