高良姜素通过PI3K/Akt及p38-MAPK信号通路增强胃癌SGC-7901细胞对阿帕替尼的敏感性

摘要：目的 探讨高良姜素对阿帕替尼抗胃癌作用的影响及可能机制。方法 体外培养胃癌SGC-7901细胞, 3-（4,5-二甲基噻唑-2）-2,5-二苯基四氮唑溴盐（MTT）法筛选出高良姜素和阿帕替尼抑制胃癌细胞增殖的合适浓 度。将胃癌细胞分为4组：空白对照组、高良姜素组、阿帕替尼组和高良姜素+阿帕替尼组。MTT法检测细胞增殖活 力;流式细胞术检测细胞凋亡率;细胞划痕实验和Transwell小室实验分别检测细胞迁移和侵袭能力;Western blot检 测细胞凋亡及PI3K/Akt及p38-MAPK信号通路相关蛋白表达。结果 阿帕替尼和高良姜素均显著抑制胃癌SGC- 7901细胞的增殖,并呈浓度依赖性（P＜0.05）,20 mg/L阿帕替尼和300 mg/L高良姜素是最佳的干预浓度。与20 mg/L 阿帕替尼组相比,高良姜素+阿帕替尼组可以明显抑制胃癌SGC-7901细胞的增殖、迁移和侵袭（P＜0.05）。流式细 胞术结果显示,高良姜素+阿帕替尼组对胃癌SGC-7901细胞促凋亡作用明显优于20 mg/L阿帕替尼组（P＜0.05）。 Western blot结果显示,高良姜素+阿帕替尼组Bcl-2相关X蛋白（Bax）、磷酸化蛋白激酶B（p-Akt）、磷酸化丝裂原活 化蛋白激酶（p-p38）蛋白表达明显高于20 mg/L阿帕替尼组,B淋巴细胞瘤-2（Bcl-2）蛋白明显低于20 mg/L阿帕替尼 组（P＜0.05）。结论 高良姜素可能通过抑制PI3K/Akt和激活p38-MAPK信号通路增强阿帕替尼抗胃癌SGC-7901 细胞的作用。     

Ultrafine particles of Ulmus davidiana var. japonica induce apoptosis of gastric cancer cells via activation of caspase and endoplasmic reticulum stress

Small-sized particles are more suitable for targeted delivery and are therapeutically more effective than large-sized particles. In this study, we investigated the anticancer effects of ultrafine particles of Ulmus davidiana var. japonica (ufUJ) on human gastric cancer cell lines SNU-1, SNU-216, and SNU-484. ufUJ induced apoptosis by the proteolytic activation of caspase-9, caspase-6, and caspase-3 and cleavage of poly (ADP-ribose) polymerase. The expression levels of the endoplasmic reticulum stress-related protein BiP markedly increased after ufUJ treatment. BiP knockdown decreased ufUJ-induced cell death. ufUJ-induced apoptosis was inhibited by the caspase-3 inhibitor z-DEVD-fmk, caspase-6 inhibitor z-VEID-fmk, and caspase-9 inhibitor z-LEHD-fmk, and by siRNAs against caspases 3, 6, and 9. Gastric cancer cells did not show anchorage-independent growth in the presence of ufUJ. However, cells treated with caspase inhibitors showed an enhanced colony-forming ability. These findings may be helpful in the prevention of gastric cancer and in the development of functional foods.     

Danthron, an anthraquinone derivative, induces DNA damage and caspase cascades-mediated apoptosis in SNU-1 human gastric cancer cells through mitochondrial permeability transition pores and Bax-triggered pathways

Anthraquinones have been shown to induce apoptosis in different types of tumor cells, but the mechanisms of danthron-induced cytotoxicity and apoptosis in human gastric cancer cells have not been adequately explored. This study investigated the roles of caspase cascades, ROS, DNA damage, mitochondrial disruption, and Bax and Bcl-2 proteins in danthron-induced apoptosis of SNU-1 human gastric cancer cells, a commonly used cell culture system for in vitro studies. Cells were incubated with different concentrations of danthron in a time- and/or dose-dependent manner. Cell morphological changes (shrinkage and rounding) were examined by a phase-contrast microscope, whereas cell viability and apoptotic populations were determined by flow cytometric analysis using propidium iodide (PI) and annexin V-FITC staining. The fluorescent DAPI nucleic acid stain and Comet assay were applied to detect danthron-induced chromatin condensation (an apoptotic characteristic) and DNA damage. Increasing the levels of caspase-3, -8, and -9 activities was involved in danthron-induced apoptosis, and they could be attenuated by inhibitors of specific caspases, indicating that danthron triggered the caspase-dependent apoptotic pathway. Further studies with flow cytometric analyses indicated that cellular levels of ROS, cytosolic Ca(2+), and mitochondrial permeability transition (MPT) pore opening were increased, but the level of mitochondrial membrane potential (ΔΨ(m)) was decreased. Also, the ratio of Bax/Bcl-2 levels and other proapoptotic proteins associated with modulating the ΔΨ(m) were up-regulated. Apoptotic signaling was also stimulated after exposure to danthron and determined by Western blotting and real-time PCR analyses. In summary, it is suggested that danthron-induced apoptotic cell death was involved in mitochondrial depolarization, which led to release of cytochrome c, apoptosis-inducing factor (AIF), and endonuclease G (Endo G) and caused the activation of caspase-9 and -3 in SNU-1 human gastric cancer cells.     

Galangin (3,5,7-Trihydroxyflavone) Shields Human Keratinocytes from Ultraviolet B-Induced Oxidative Stress

Most skin damage caused by ultraviolet B (UVB) radiation is owing to the generation of reactive oxygen species. Phytochemicals can act as antioxidants against UVB-induced oxidative stress. This study investigated the protective effects of the flavone galangin against UVB-induced oxidative damage in human keratinocytes. Galangin efficiently scavenged free radicals and reduced UVB-induced damage to cellular macromolecules, such as DNA, lipids, and proteins. Furthermore, galangin rescued cells undergoing apoptosis induced by UVB radiation via recovering mitochondrial polarization and down-regulating apoptotic proteins. These results showed that galangin protects human keratinocytes against UVB radiation-induced cellular damage and apoptosis via its antioxidant effects.     

The Effect of Galangin on the Regulation of Vascular Contractility via the Holoenzyme Reactivation Suppressing ROCK/CPI-17 rather than PKC/CPI-17

In this study, we investigated the influence of galangin on vascular contractibility and to determine the mechanism underlying the relaxation. Isometric contractions of denuded aortic muscles were recorded and combined with western blot analysis which was performed to measure the phosphorylation of phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17) and myosin phosphatase targeting subunit 1 (MYPT1) and to evaluate the effect of galangin on the RhoA/ROCK/CPI-17 pathway. Galangin significantly inhibited phorbol ester-, fluoride- and thromboxane mimetic-induced vasoconstrictions regardless of endothelial nitric oxide synthesis, suggesting its direct effect on vascular smooth muscle. Galangin significantly inhibited the fluoride-dependent increase in pMYPT1 and pCPI-17 levels and phorbol 12,13-dibutyrate-dependent increase in pERK1/2 level, suggesting repression of ROCK and MEK activity and subsequent phosphorylation of MYPT1, CPI-17 and ERK1/2. Taken together, these results suggest that galangin-induced relaxation involves myosin phosphatase reactivation and calcium desensitization, which appears to be mediated by CPI-17 dephosphorylation via not PKC but ROCK inactivation.     

Involvement of Kv4.1 K(+) channels in gastric cancer cell proliferation

Voltage-gated potassium (Kv) channels are expressed not only in excitable cells but also in non-excitable cells such as epithelial cells. Recent studies have demonstrated that several subtypes of Kv channels are expressed in epithelial tumor cells, including human gastric cancer cells, and are associated with cell proliferation. In the present study, we examined the expression of Kv4.1 in human gastric cancer cell lines and the effects of suppressed expression of Kv4.1 on cell proliferation and cell cycle distribution. We found that Kv4.1 mRNA and protein are expressed in the human gastric cancer cell lines MKN-45 and SNU-638. Moreover, Kv4.1-targeted small interference RNA (siRNA) treatment inhibited gastric cancer cell proliferation. Flow cytometric analysis revealed that suppressed expression of Kv4.1 induced a G1-S transition block of cell cycle progression. These results reveal that Kv4.1 plays a role in the proliferation of the human gastric cancer cell lines MKN-45 and SNU-638 and can be considered as a therapeutic target for human gastric cancer.     

Galangin suppresses H2O2‐induced aging in human dermal fibroblasts

Human skin aging is a progressive process that includes intrinsic aging and extrinsic photodamage, both of which can cause an accumulation of reactive oxygen species (ROS), resulting in dermal fibrosis dysfunction and wrinkle formation. Galangin is a flavonoid that exhibits anti‐inflammatory and antioxidative potential. Previous studies have reported that galangin has antioxidative activity against ROS‐mediated stress. The aim of the present study is to determine the antiaging effects of galangin on dermal fibroblasts exposed to H₂O₂. In this study, we established a hydrogen peroxide‐induced inflammation and aging model using human HS68 dermal fibroblasts. Stimulation of fibroblasts with H₂O₂ is associated with skin aging and increased expression of inflammation‐related proteins, along with downregulation of collagen I/III formation and expression of antioxidative proteins. Galangin effectively reduced NF‐κB activation, the expression of inflammation‐related proteins and cell aging. Galangin also reversed H₂O₂‐activated cell senescence in HS68 cells. Our results reveal that galangin protects human dermal fibroblasts by inhibiting NF‐κB activation, decreases the expression of inflammatory factors and upregulates IGF1R/Akt‐related proteins, indicating that galangin may be a potential candidate for developing natural antiaging products that protect skin from damage caused by ROS.     

Berberine induced down-regulation of matrix metalloproteinase-1, -2 and -9 in human gastric cancer cells (SNU-5) in vitro

Berberine, a yellow benzylisoquinoline alkaloid, is a constituent of Coptis chines and is commonly used in Chinese herbal medicine for patients with gastrointestinal disorders. The pharmacological effects of berberine include anti-inflammation, antidiarrhetic, antimalarial, and even antimicrobial activities. However, its mechanism of action on the cell migration of human gastric cancer SNU-5 cells is not fully understood. The effects of berberine on the percentage of viable cells were examined first and it was found that berberine induced dose-dependent inhibition in human gastric cancer SNU-5 cells. The effect of berberine on the levels of reactive oxygen species (ROS) and matrix metalloproteinase-1, -2, -7 and -9 was then examined using Western blotting and the results showed that berberine induced ROS production for up to 6 hours of incubation. It was also found that berberine induced downregulation of MMP-1 -2, and -9 but did not affect the level of MMP-7. The mRNA levels of MMPs in SNU-5 cells after treatment with berberine for 24 hours were investigated using a polymerase chain reaction and the results showed that berberine inhibited the gene expression of MMP-1, -2 and -9 in human SNU-5 cells but it did not affect MMP-7. In conclusion, berberine appears to exert its anticancer properties by inducing ROS production and prevention of cell migration via inhibition of the gene expression of MMP-1, -2 and -9 in human gastric cancer SNU-5 cancer cells.     

Arctigenin induces cell cycle arrest by blocking the phosphorylation of Rb via the modulation of cell cycle regulatory proteins in human gastric cancer cells

Gastric cancer is a leading cause of cancer-related deaths, worldwide being second only to lung cancer as a cause of death. Arctigenin, a representative dibenzylbutyrolactone lignan, occurs in a variety of plants. However, the molecular mechanisms of arctigenin for anti-tumor effect on gastric cancer have not been examined. This study examined the biological effects of arctigenin on the human gastric cancer cell line SNU-1 and AGS. Cell proliferation was determined by MTT assay. In MTT assay, the proliferation of SNU-1 and AGS cells was significantly inhibited by arctigenin in a time and dose dependent manner, as compared with SNU-1 and AGS cells cultured in the absence of arctigenin. Inhibition of cell proliferation by arctigenin was in part associated with apoptotic cell death, as shown by changes in the expression ratio of Bcl-2 to Bax by arctigenin. Also, arctigenin blocked cell cycle arrest from G₁ to S phase by regulating the expression of cell cycle regulatory proteins such as Rb, cyclin D1, cyclin E, CDK4, CDK2, p21Waf1/Cip1 and p15 INK4b. The antiproliferative effect of arctigenin on SNU-1 and AGS gastric cancer cells revealed in this study suggests that arctigenin has intriguing potential as a chemopreventive or chemotherapeutic agent.     

Galangin induces apoptosis in hepatocellular carcinoma cells through the caspase 8/t-Bid mitochondrial pathway

This study has investigated whether galangin, a flavonol derived from Alpinia officinarum Hance and used as food additives in southern China, induces apoptosis in hepatocellular carcinoma cells (HCCs) by activation of the caspase-8 and Bid pathway. The apoptosis of HCCs was evaluated by in situ uptake of propidium iodide and Hoechst 33258. Protein expressions were detected by Western blotting. Caspase-8 activity was measured using colorimetric method. To confirm the galangin-induced apoptotic pathway, inhibition of caspase-8 activity by Z-IETD-FMK, knockdown of Bid expression with siRNA, and overexpression of Bcl-2 in cells were carried out, respectively. The results show that galangin has significantly induced apoptosis in HCC lines. The caspase-8 is activated, and the cleavage of Bid results in the increase in tBid. The galangin-induced apoptosis is attenuated by Z-IETD-FMK, Bid siRNA, and Bcl-2 overexpression, respectively. However, Bcl-2 fails to suppress caspase-8 activation and the cleavage of Bid. This study has demonstrated that galangin induces apoptosis in HCCs by activating caspase 8/t-Bid mitochondrial pathway. Although Bcl-2 overexpression attenuates galangin-mediated apoptosis of HCCs, it is not mediated by the inhibition of tBid generation and caspase-8 activation.     

Ganciclovir augments the lytic induction and apoptosis induced by chemotherapeutic agents in an Epstein-Barr virus-infected gastric carcinoma cell line

Epstein-Barr virus is an oncogenic herpesvirus and has been associated with several human malignancies, including gastric cancer. In Epstein-Barr virus-associated gastric cancer, Epstein-Barr virus is found in virtually all tumor cells, but rarely in normal epithelial cells, thus implying that Epstein-Barr virus-targeting therapies are likely to be an effective treatment strategy. Using the SNU-719 gastric cancer cell line, which is naturally infected with Epstein-Barr virus, we found that the chemotherapeutic agents, such as 5-fluorouracil, cis-platinum and taxol, induced the expressions of BMRF1, BZLF1 and BRLF1 proteins that are usually found in the lytic form of the virus. This effect was found to require various signal transduction pathways involving phosphatidylinositol 3' kinase, mitogen-activated protein/extracellular signal-regulated kinase, protein kinase C delta and p38 mitogen-activated protein kinase. Moreover, the combination of ganciclovir with these agents increased the lytic transformation and induced apoptosis in Epstein-Barr virus-associated gastric carcinoma. We conclude that ganciclovir enhances the therapeutic efficacy of 5-fluorouracil, cis-platinum and taxol in Epstein-Barr virus-positive gastric cancer cells. It is hoped that this information will be found useful during the establishment of treatment strategies for Epstein-Barr virus-associated gastric cancer.     

Galangin induces apoptosis in hepatocellular carcinoma cells through the caspase 8/t-Bid mitochondrial pathway

This study has investigated whether galangin, a flavonol derived from Alpinia officinarum Hance and used as food additives in southern China, induces apoptosis in hepatocellular carcinoma cells (HCCs) by activation of the caspase-8 and Bid pathway. The apoptosis of HCCs was evaluated by in situ uptake of propidium iodide and Hoechst 33258. Protein expressions were detected by Western blotting. Caspase-8 activity was measured using colorimetric method. To confirm the galangin-induced apoptotic pathway, inhibition of caspase-8 activity by Z-IETD-FMK, knockdown of Bid expression with siRNA, and overexpression of Bcl-2 in cells were carried out, respectively. The results show that galangin has significantly induced apoptosis in HCC lines. The caspase-8 is activated, and the cleavage of Bid results in the increase in tBid. The galangin-induced apoptosis is attenuated by Z-IETD-FMK, Bid siRNA, and Bcl-2 overexpression, respectively. However, Bcl-2 fails to suppress caspase-8 activation and the cleavage of Bid. This study has demonstrated that galangin induces apoptosis in HCCs by activating caspase 8/t-Bid mitochondrial pathway. Although Bcl-2 overexpression attenuates galangin-mediated apoptosis of HCCs, it is not mediated by the inhibition of tBid generation and caspase-8 activation.     

miR-578调控长链脂酰辅酶A合成酶4表达对胃癌细胞增殖、侵袭的影响

目的 探讨miR-578调控长链脂酰辅酶A合成酶4（ACSL4）对胃癌细胞增殖及侵袭的影响。方法 选取人胃癌细胞MKN-45、SNU-1、MKN-7、KTAO3、N87及人正常胃黏膜细胞GES-1为研究对象,将MKN-45细胞分为对照组、miR-578 NC组、miR-578 mimic组、miR-578 mimic+pc-ACSL4组,测定各组MKN-45细胞活力及凋亡、侵袭、迁移能力,测定细胞中miR-578、ACSL4、凋亡相关蛋白（Bax、Caspase-3、Bcl-2）、侵袭相关蛋白（MMP-9、MMP-2）的表达,验证miR-578与ACSL4的靶向关系。结果 miR-578在人胃癌细胞中低表达;与对照组相比,miR-578 mimic组MKN-45细胞活力、侵袭、迁移能力、MMP-2、MMP-9、Bcl-2蛋白表达水平均显著降低（P<0.05）,凋亡能力、Caspase-3、Bax蛋白表达水平均显著升高（P<0.05）;与miR-578 mimic组相比,miR-578 mimic+pc-ACSL4组MKN-45细胞活力、侵袭、迁移能力、MMP-2、MMP-9、Bcl-2蛋白表达水平均显著升高（P<0.05）,凋亡能力、Caspase-3、Bax蛋白表达水平均显著降低（P<0.05）;荧光素酶实验结果显示,miR-578与ACSL4存在靶向调节关系。结论 miR-578可抑制人胃癌细胞MKN-45的增殖及迁移能力,可能是通过抑制ACSL4表达实现的。     

Synergistic interaction between gefitinib (Iressa, ZD1839) and paclitaxel against human gastric carcinoma cells

We have evaluated the antitumor effects of gefitinib (Iressa, ZD1839) in SNU-1 human gastric cancer cells (hMLH1-deficient and epidermal growth factor receptor-overexpressed) when given alone or as a doublet with oxaliplatin (LOHP), 5-fluorouracil (5-FU) or paclitaxel (PTX). The four drugs showed IC50s ranging from 1.81 nM to 13.2 microM. LOHP and PTX induced G2/M arrest, 5-FU increased S phase, and gefitinib increased G1 in a concentration-dependent manner. The analysis using the previously developed cytostatic TPi model showed that 64 and 80% of the overall growth inhibition was attributed to cell cycle arrest in cells exposed to 7.55 microM of LOHP or 10 nM of PTX for 72 h, respectively. PTX + gefitinib showed greatest synergism as determined by combination index analysis and apoptosis induced by PTX was potentiated by the co-administration of gefitinib. LOHP + gefitinib showed a similar, although to a lesser degree, synergistic effect. This study demonstrates the antitumor activity and the significant cell cycle arrest induced by gefitinib in SNU-1 human gastric carcinoma cells, and its synergistic interaction with LOHP and PTX.     

Topoisomerase I inhibitor (camptothecin)-induced apoptosis in human gastric cancer cells and the role of wild-type p53 in the enhancement of its cytotoxicity

Camptothecin (CPT), a human topoisomerase I inhibitor, blocks DNA replication in human cancer cells. It represents a promising new class of chemotherapeutic agents with broad anti-tumor activity. However, its effect on gastric cancer cells remains unknown. We examined cell growth, apoptosis and cell cycle phase distribution in gastric cancer cells by exposing these cells to CPT for up to 72 h. Cell viability was determined by the Trypan blue exclusion assay. Cell cycle phase distribution and apoptosis were measured using flow cytometry, fluorescence microscopy and DNA ladder assay. Exposure of exponentially growing gastric AGS cancer cells to CPT induced time-dependent apoptosis and growth inhibition. Serum starvation-synchronized AGS cells (about 60% cells in G0/G1 phase) showed similar cellular responses. Analysis of cell cycle phase distribution of AGS cells treated with CPT for up to 72 h showed no obvious differences compared to untreated control cells. Although the induction of apoptosis was noticed in gastric cancer cell lines both with and without p53, cells lacking p53 showed less apoptosis compared to those cell lines possessing p53. Our data show that CPT is capable of inducing gastric cancer cell growth inhibition and apoptosis. Wild-type p53 may enhance the cytotoxicity of CPT against gastric carcinoma.     

Inhibitory effect of galangin on atopic dermatitis-like skin lesions

Galangin is a member of the flavonol class of flavonoids having anti-inflammatory and anti-oxidative potential. Previously we reported the inhibitory effect of galangin on the mast cell-mediated allergic inflammation. For incremental research, we investigated the effects of galangin on atopic dermatitis (AD)-like skin lesions and underlying mechanisms of action. We established an atopic dermatitis model in BALB/c mice by repeated local exposure of house dust mite (Dermatophagoides farinae) extract (DFE) and 2,4-dinitrochlorobenzene (DNCB) to the ears. Repeated alternative treatment of DFE/DNCB caused AD-like skin lesions. Topical application of galangin reduced AD symptoms based on ear thickness and histopathological analysis, in addition to serum IgE and IgG2a levels. Galangin inhibited mast cell infiltration into the ear and serum histamine level. Galangin suppressed DFE/DNCB-induced expression of interleukin (IL)-4, IL-5, IL-13, IL-31, IL-32, and interferon (IFN)-γ in the ear tissue. To define the underlying mechanisms of action, tumor necrosis factor-α/IFN-γ-activated human keratinocytes (HaCaT) model was used. Galangin significantly inhibited the expression of cytokines and chemokine by the down-regulation of nuclear factor-κB and mitogen-activated protein kinases in HaCaT cells. Taken together, the results demonstrate that galangin inhibited AD-like symptoms, suggesting that galangin might be a candidate for the treatment of AD.     

Synthesis andin vitro antitumor activity of 2-alkyl, 2-aryl, and 2-piperazinyl benzimidazole-4,7-dione derivatives

A series of 2-alkyl, 2-aryl, and 2-piperazinyl benzimidazole-4,7-dione derivatives (7a-h) and 16m-o) were prepared, and their cytotoxicities were tested against three cancer cell lines (mouse lymphocytic leukemia cell line P388, and human gastric carcinoma cell lines SNU-1 and SNU-16). These compounds showed potent cytotoxicity against all of three cell lines tested, and especially SNU-16 was sensitive to them. 2-Aryl (7g,h) and 2-piperazinyl benzimidazole-4,7-dione derivative (16 m) were more potent than mitomycin C against P388 and SNU-16. Among benzimidazole-4,7-dione derivatives with alkyl group at position 2, 7a had the most potent cytotoxicity against all of the cell lines tested.     

GSK461364A suppresses proliferation of gastric cancer cells and induces apoptosis

Polo-like kinase 1 (PLK1) is crucial in regulating cell division and has been shown to have an oncogenic function in several cancers. Since PLK1 overexpression is closely related to tumorigenesis and has been correlated with poor clinical outcomes, specific inhibition of PLK1 in cancer cells is a promising approach for developing new anticancer drugs. In this context, the aim of the present study was to evaluated the potential cytotoxic effects of GSK461364A, a competitive inhibitor for PLK1, in gastric cancer cell line SNU-1 cells and explored its cytotoxic mechanism. The cells were exposed to GSK461364A at different concentrations ranging from 1 to 40 μM for 24 h, and it showed considerable cytotoxicity with an IC₅₀ value of 4.34 μM. The treatment of SNU-1 cells with GSK461364A results in cell cycle arrest at the G2/M phase, decreased mitochondrial membrane potential, and increased apoptosis as indicated by Annexin V binding assay. In addition, GSK461364A treatment significantly increased the total oxidant (TOS) level, a signal of oxidative stress, and increased cleaved PARP and 8-oxo-dG levels as an indicator of DNA damage. ELISA experiments evaluating Bax, BCL-2, and cleaved caspase 3 also confirmed the apoptotic effect of GSK461364A. Current findings suggest that GSK461364A may be a chemotherapeutic agent in patients with gastric cancer. Nevertheless, more research is needed to evaluate GSK461364A as a cancer treatment drug.