Osthole induces G2/M arrest and apoptosis in lung cancer A549 cells by modulating PI3K/Akt pathway

Background

To explore the effects of Osthole on the proliferation, cell cycle and apoptosis of human lung cancer A549 cells.

Methods

Human lung cancer A549 cells were treated with Osthole at different concentrations. Cell proliferation was measured using the MTT assay. Cell cycle was evaluated using DNA flow cytometry analysis. Induction of apoptosis was determined by flow cytometry and fluorescent microscopy. The expressions of Cyclin B1, p-Cdc2, Bcl-2, Bax, t-Akt and p-Akt were evaluated by Western blotting.

Results

Osthole inhibited the growth of human lung cancer A549 cells by inducing G2/M arrest and apoptosis. Western blotting demonstrated that Osthole down-regulated the expressions of Cyclin B1, p-Cdc2 and Bcl-2 and up-regulated the expressions of Bax in A549 cells. Inhibition of PI3K/Akt signaling pathway was also observed after treating A549 cells with Osthole.

Conclusions

Our findings suggest that Osthole may have a therapeutic application in the treatment of human lung cancer.     

Cochinchina momordica seed extract induces G2/M arrest and apoptosis in human breast cancer MDA-MB-231 cells by modulating the PI3K/Akt pathway

Cochinchina momordica seeds are a kind of traditional Chinese herb. In this study, anticancer activity and underlying mechanisms were investigated with an extract using human breast cancer MDA-MB-231 cells. The survival rate was reduced in a concentration- and time-dependent manner as assessed by MTT assay. After incubation for 48 h, typical apoptotic morphological changes were observed by Hoechst 33258 dye assay. Flow cytometry revealed that the treatment obviously induced G2/M arrest and apoptosis in MDA-MB-231 cells. Furthermore, western blotting demonstrated downregulation of protein expression of PI3K, Akt, NF-kB, Bcl-2, Cdk1 and cyclin B1, whereas Bax and caspase-3 were upregulated. Our results suggest that the extract induced cell cycle G2/M arrest and apoptosis in MDA-MB-231 cells by decreasing PI3K/Akt pathway. Therefore, we propose that ECMS has potential as a breast cancer chemotherapeutic agent.     

beta-Sitosterol induces G2/M arrest, endoreduplication, and apoptosis through the Bcl-2 and PI3K/Akt signaling pathways

β-Sitosterol (SITO) is a potentially valuable candidate for cancer chemotherapy, however the cellular and molecular mechanisms responsible for its anti-cancer activity are unknown. Therefore, we attempted to elucidate the mechanisms responsible for SITO-induced anti-proliferation in human leukemia cells. Treatment with SITO increased caspase-3 activation and DNA fragmentation in U937 and HL60 cells. This effect was associated with significant G₂/M arrest and endoreduplication. We also demonstrated that SITO treatment significantly increases levels of polymeric -tubulin and promoted microtubule polymerization. We next elucidated that ectopic expression of Bcl-2 accelerates endoreduplication in U937 cells. Furthermore, the specific Bcl-2 inhibitor, HA14-1, prevented endoreduplication through G₂ phase arrest. Interestingly, SITO treatment did not significantly promote endoreduplication or decrease cell viability in Bcl-2 null K562 cells. SITO treatment also induced a gradual increase of phosphatidyl-inositol 3-kinase (PI3K) and Akt phosphorylation. Treatment with the selective PI3K/Akt inhibitor LY29004 completely blocked endoreduplication and apoptosis in the presence of SITO. In addition, treatment with SITO-induced phosphorylation of extracellular signal-regulated protein kinase (ERK), however significance of ERK activation in the execution of apoptosis and endoreduplication is unknown. These results suggest that SITO induces endoreduplication by promoting spindle microtubule dynamics through the Bcl-2 and PI3K/Akt signaling pathways.     

芹菜素通过 PI3K/Akt 信号通路诱导 Hela 细胞凋亡

目的：初探芹菜素（Apigenin）对人宫颈癌细胞 Hela 的抑制机制。方法：MTT 法检测 Apigenin 对 Hela细胞的增殖抑制作用,实时无标记细胞分析技术测量 Apigenin 对 Hela 细胞生长曲线的影响。Western blot 检测 Apigenin 对 Hela 细胞中 PI3K/ Akt 信号通路的影响。结果：与阴性对照组相比,Apigenin 能显著抑制 Hela细胞的增殖（P <0.01）,当药物浓度为20μmol/ L 时抑制率达到（80.5±5.3）%,IC50值为（6.8±0.8）μmol/ L。细胞生长曲线反映,Apigenin 减缓了 Hela 细胞的增殖,对数期细胞经20μmol/ L Apigenin 处理后,细胞数量迅速下降。Western blot 结果显示,Apigenin 能显著抵抗 IGF -1引起的 Akt 激活但并不能降低 PI3K 的磷酸化水平。同时 Apigenin 还能降低 Bcl -2/ Bax 比例,提高 Caspase -3活性,启动细胞线粒体凋亡。结论：Apigenin可以通过 PI3K/ Akt 信号通路促进 Hela 细胞的凋亡。     

红花多糖抑制PI3K/Akt信号通路诱导人胃癌细胞凋亡的研究

目的 探讨红花多糖通过抑制PI3K/Akt信号通路调控胃癌细胞凋亡的机制。方法 MTT比色法观察SPS对人胃癌SGC-7901细胞体外增殖的抑制作用,流式细胞仪(FCM)分析细胞凋亡,实时荧光定量RT-PCR法和Western Blot法检测蛋白激酶B(Akt)基因及蛋白的表达情况。结果 红花多糖在一定范围内以剂量依赖方式和时间依赖方式抑制SGC-7901胃癌细胞生长。流式细胞仪检测,SGC-7901细胞经红花多糖处理24 h,其早期凋亡率、细胞坏死或晚期凋亡率显著增加,呈现明显的剂量依赖性。Real-time PCR和Western Blot检测发现,SPS处理的细胞Akt基因及蛋白表达量明显下降。结论 红花多糖对人胃癌SGC-7901细胞体外增殖具有明显的抑制作用,该抑制作用具有一定的时间依赖性和剂量依赖性;红花多糖能够下调Akt mRNA表达,降低Akt和p-Akt蛋白的表达量,抑制Akt通路发挥抗肿瘤作用。     

Curcumin induces G2/M arrest and apoptosis in cisplatin-resistant human ovarian cancer cells by modulating Akt and p38 MAPK

Curcumin, a major active component of turmeric, is known to induce apoptosis in several types of cancer cells, but little is known about its activity in chemoresistant cells. Hence, the aim of the present study was to investigate the anticancer properties of curcumin in cisplatin-resistant human ovarian cancer cells in vitro. The results indicated that curcumin inhibited the proliferation of both cisplatin-resistant (CR) and sensitive (CS) human ovarian cancer cells almost equally. Enhanced superoxide generation was observed in both CR and CS cells treated with curcumin. Curcumin induced G(2)/M phase cell-cycle arrest in CR cells by enhancing the p53 phosphorylation and apoptosis through the activation of caspase-3 followed by PARP degradation. Curcumin also inhibited the phosphorylation of Akt while the phosphorylation of p38 MAPK was enhanced. In summary, our results showed that curcumin inhibits the proliferation of cisplatin-resistant ovarian cancer cells through the induction of superoxide generation, G(2)/M arrest, and apoptosis.     

M2-A induces apoptosis and G2–M arrest via inhibiting PI3 K/Akt pathway in HL60 cells

Amonafide, a naphthalimide derivative, although selected for exploratory clinical trials for its potent anticancer activity, has long been challenged by its unpredictable side effects. In the present study, a novel amonafide analogue, M₂-A 2-(2-(dimethylamino)ethyl)-6-(thiophene-2-ylmethylamino)-1H-benzo[de]isoquinoline-1,3(2H)-dione was ascribed to its potent effects on topoisomerase IIα. Moreover, our investigation indicates that M₂-A induces G₂/M phase growth arrest through inhibiting PI3 K/Akt pathway. M₂-A inhibits proliferation of HeLa, HL60, HCT-8, A375, MCF-7 and MRC-5 cells, especially inhibits proliferation of HL60 with an IC₅₀ value of 18.86 μM. M₂-A can not only induce DNA fragmentation, but also enhance Annexin V-FITC binding of the cells. On the one hand the expression levels of protein Cyclin B1, Cdk1 changed in response to M₂-A treatment in HL60 cells. On the other hand we observed the inhibition of NF-κB nuclear translocation, up-regulation of Bax and down-regulation of Bcl-2, the caspase -3, -9 activity increase in HL60 cells after treated with M₂-A, which indicated that the mitochondrial pathway was involved in the apoptosis signal pathway. Our results showed that the phosphorylation of p85/PI3 K and Akt decreased following M₂-A treatment. In summary, M₂-A displayed a significant anti-tumor effect through cell cycle arrest and apoptotic induction in HL60 cells, which suggested that M₂-A might have therapeutic potential against leukaemia.     

Oxymatrine induces mitochondria dependent apoptosis in human osteosarcoma MNNG/HOS cells through inhibition of PI3K/Akt pathway

The cytostatic drug from traditional Chinese medicinal herb has acted as a chemotherapeutic agent used in treatment of a wide variety of cancers. Oxymatrine, classified as a quinolizidine alkaloid, is a phytochemical product derived from Sophora flavescens, and has been reported to possess anticancer activities. However, the cancer growth inhibitory effects and molecular mechanisms in human osteosarcoma MNNG/HOS cell have not been well studied. In the present study, the cytotoxic effects of oxymatrine on MNNG/HOS cells were examined by MTT and bromodeoxyuridine (BrdU) incorporation assays. The percentage of apoptotic cells and the level of mitochondrial membrane potential (Δψ _m) were assayed by flow cytometry. The levels of apoptosis-related proteins were measured by Western blot analysis or enzyme assay Kit. Our results showed that treatment with oxymatrine resulted in a significant inhibition of cell proliferation and DNA synthesis in a dose-dependent manner, which has been attributed to apoptosis. Furthermore, we found that oxymatrine considerably inhibited the expression of Bcl-2 whilst increasing that of Bax. This promoted mitochondrial dysfunction, leading to the release of cytochrome c from the mitochondria to the cytoplasm, as well as the activation of caspase-9 and -3. Moreover, addition of oxymatrine to MNNG/HOS cells also attenuated phosphatidylinositol 3-kinase (PI3K) ?Akt signaling pathway cascade, evidenced by the dephosphorylation of P13K and Akt. Likewise, oxymatrine significantly suppressed tumor growth in female BALB/C nude mice bearing MNNG/HOS xenograft tumors. In addition, no evidence of drug-related toxicity was identified in the treated animals by comparing the body weight increase and mortality. Therefore, these findings should be useful for understanding the apoptotic cellular mechanism mediated by oxymatrine and might offer a therapeutic potential advantage for human osteosarcoma chemoprevention or chemotherapy.     

Thimerosal induces apoptosis and G2/M phase arrest in human leukemia cells

Thimerosal is an organomercury compound with sulfhydryl-reactive properties. The ability of thimerosal to act as a sulfhydryl group is related to the presence of mercury. Due to its antibacterial effect, thimerosal is widely used as preservatives and has been reported to cause chemically mediated side effects. In the present study, we showed that the molecular mechanism of thimerosal induced apoptosis in U937 cells. Thimerosal was shown to be responsible for the inhibition of U937 cells growth by inducing apoptosis. Treatment with 2.5-5 microM thimerosal but not thiosalicylic acid (structural analog of thimerosal devoid of mercury) for 12 h produced apoptosis, G(2)/M phase arrest, and DNA fragmentation in a dose-dependent manner. Treatment with caspase inhibitor significantly reduced thimerosal-induced caspase 3 activation. In addition, thimerosal-induced apoptosis was attenuated by antioxidant Mn (III) meso-tetrakis (4-benzoic acid) porphyrin (Mn-TBAP). These data indicate that the cytotoxic effect of thimerosal on U937 cells is attributable to the induced apoptosis and that thimerosal-induced apoptosis is mediated by reactive oxygen species generation and caspase-3 activation.     

Methyl protodioscin induces G2/M cell cycle arrest and apoptosis in HepG2 liver cancer cells

Methyl protodioscin (NSC-698790) is one of the main bioactive components in the traditional Chinese medicine Dioscorea collettii var. hypoglauca (Dioscoreaceae). In this study, we investigated the anti-proliferative effect of methyl protodioscin on the HepG2 cells and the mechanism of the induced cytotoxicity. Treatment of methyl protodioscin resulted in G2/M arrest and apoptosis in HepG2 cells. These effects were attributed to down-regulation of Cyclin B1 and the signaling pathways leading to up-regulation of Bax and down-regulation of BCL2, suggesting that methyl protodioscin may be a novel anti-mitotic agent.     

贝母素乙通过调控PI3K/Akt信号通路诱导乳腺癌MCF-7细胞凋亡

目的：探究贝母素乙（Peiminine）对乳腺癌细胞MCF-7细胞凋亡的影响及其可能作用机制。方法：采用不同浓度贝母素乙或联合PI3K抑制剂LY294002干预MCF-7细胞,MTT法检测细胞增殖能力;Hoechst33258染色和Annexin V-FITC/PI流式细胞术检测细胞凋亡情况;JC-1染色法检测细胞线粒体膜电位变化;Western blotting检测细胞中PI3K(p110α)、Akt、p-Akt(ser473)、Bad、Bax、Bcl-2、cleaved-Caspase-3以及线粒体和胞浆中细胞色素C(Cyt C)等蛋白表达水平。结果：贝母素乙可呈时间-浓度依赖性抑制MCF-7细胞增殖,诱导细胞出现凋亡形态改变,促进细胞凋亡,并降低线粒体膜电位,上调细胞中Bad、Bax、cleaved-Caspase-3及胞浆中Cyt C蛋白表达水平,下调PI3K(p110α)、p-Akt、Bcl-2和线粒体中Cyt C蛋白表达水平,而Akt蛋白表达水平无显著变化。然而,联合LY294002干预可增强贝母素乙对MCF-7细胞凋亡的促进作用。结论：贝母素乙可诱导乳腺癌MCF-7细胞凋...     

Arsenic trioxide induces apoptosis and G2/M phase arrest by inducing Cbl to inhibit PI3K/Akt signaling and thereby regulate p53 activation

Arsenic trioxide (ATO) strongly induces apoptosis in acute promyelocytic leukemia (APL), but it induces cell cycle arrest in most solid tumors. In this study, we investigated the mechanism of ATO action on APL-derived NB4 cells and gastric cancer cell lines. ATO decreased the viability of both cell lines, but gastric cancer cells were much less susceptible. ATO-induced G2/M phase arrest and p53 degradation in gastric cancer MGC803 cells. In contrast, ATO-induced apoptosis in NB4 cells without degradation of p53. Both processes were accompanied by transient activation of Akt. The PI3K/Akt inhibitor LY294002 significantly increased the amount of p53 protein and ATO-induced apoptosis in both cell lines and decreased G2/M phase arrest of MGC803 cells. In addition, ATO up-regulated the expression of Cbl proteins in both cell lines. Inhibition of Cbl with the proteasome inhibitor Ps341 decreased apoptosis in NB4 cells and increased the G2/M phase arrest of MGC803 cells, and it also prolonged the activation of PI3K/Akt by ATO. Consistent results with those in MGC803 cells were showed in gastric cancer cell BGC823 and SGC7901 after ATO treatment. These results demonstrate that inhibition of PI3K/Akt signaling by Cbl is involved in both ATO-induced apoptosis of NB4 cells and ATO-induced G2/M phase arrest of gastric cancer cells. Cbl achieved these effects probably via its regulating PI3K/Akt pathway, and thereby modulated p53 activation.     

Rottlerin induces autophagy and apoptosis in prostate cancer stem cells via PI3K/Akt/mTOR signaling pathway

Autophagy plays an important role in cellular homeostasis through the disposal and recycling of cellular components. Cancer stem cells (CSCs) play major roles in cancer initiation, progression, and drug resistance. Rottlerin (Rott) is an active molecule isolated from Mallotus philippinensis, a medicinal plant used in Ayurvedic Medicine for anti-allergic and anti-helminthic treatments, demonstrates anticancer activities. However, the molecular mechanisms by which it induces autophagy in prostate CSCs have not been examined. The main objective of the paper was to examine the molecular mechanisms by which Rott induces autophagy in prostate CSCs. Autophagy was measured by the lipid modification of light chain-3 (LC3) and the formation of autophagosomes. Apoptosis was measured by flow cytometer analysis. The Western blot analysis was used to examine the effects of Rott on the expression of PI3K, phosphorylation of Akt, phosphorylation of mTOR, and phosphorylation of AMPK in pros CSCs. RNAi technology was used to inhibit the expression of Beclin-1 and ATG-7. Rott induced the lipid modification of light chain-3 (LC3) and the formation of autophagosomes after 24 h of Rott treatment in prostate CSCs. Rott-treated prostate CSCs induced transition from LC3-I to LC3-II, a hall mark of autophagy. Rott also induced the expression of Atg5, Atg7, Atg12 and Beclin-1 proteins during autophagy. The knock-down of Atg7 and Beclin-1 blocked Rott-induced autophagy. Furthermore, Rott induced AMPK phosphorylation was blocked by 3-MA, Baf and CHX. In addition, inhibition of AMPK expression by shRNA blocked Rott induced autophagy. In conclusion, a better understanding of the biology of autophagy and the pharmacology of autophagy modulators has the potential for facilitating the development of autophagy-based therapeutic interventions for prostate cancer.     

Jolkinolide B induces apoptosis in MDA-MB-231 cells through inhibition of the PI3K/Akt signaling pathway

The phosphoinositol-3-kinase (PI3K)/Akt signal transduction pathway is critically important for tumor cell growth, proliferation and apoptosis. Apoptosis activation has been reported to be a good target in cancer therapies. In this study, we have found that jolkinolide?B (JB), a diterpenoid from the traditional Chinese medicinal herb Euphorbia fischeriana Steud, strongly inhibited the expression of the PI3K p85 subunit and the phosphorylation of Akt. Furthermore, we evaluated the effects of JB on the proliferation and apoptosis of MDA-MB-231 human breast cancer cells. Our results show significant induction of apoptosis in MDA-MB-231 cells incubated with JB. This effect was enhanced by combination with LY294002. In addition, treatment with JB could induce downregulation of the Bcl-2/Bax ratio, and subsequent promotion of mitochondrial release of cytochrome c and activation of caspase-3. Taken together, JB-induced apoptosis of MDA-MB-231 cells occurs through the mitochondrial pathway. Further, the PI3K/Akt signaling cascade plays a role in the induction of apoptosis in JB-treated cells. These observations suggest that JB may have therapeutic applications in the treatment of cancer.     

Blocking of PI3K/Akt pathway enhances apoptosis induced by SN-38, an active form of CPT-11, in human hepatoma cells

Hepatocellular carcinoma (HCC) is a common malignancy and often resistant to chemotherapy. Many chemotherapy regimens have been tried to control advanced HCC, but have produced a low response rate and no clear impact. CPT-11, a derivative of camptothecin, works as type-I DNA topoisomerase inhibitor and showed a major objective response rate in patients with metastatic colorectal cancer. In this study, the mechanism underlying chemo-resistance to SN-38, an active form of CPT-11, in HCC was investigated in relation to anti-apoptotic pathways NF-kappaB and PI3K/Akt. Hep3B was the most resistant to SN-38 among three hepatoma cell lines. NF-kappaB was constitutively activated in Hep3B, and SN-38 further enhanced the nuclear translocation of NF-kappaB. However, inactivation of NF-kappaB by adenovirus expressing IkappaB super-repressor or MG-132, proteasome inhibitor, did not sensitize Hep3B to SN-38-induced apoptosis. On the other hand, SN-38 phosphorylated Akt and pretreatment with PI3K inhibitors increased SN-38-induced apoptosis, indicating that resistance to SN-38 in Hep3B occurs partly through the PI3K/Akt not the NF-kappaB pathway. Blocking of PI3K/Akt may thus be helpful for overcoming chemo-resistance of HCC.     

Bicyclic triterpenoid Iripallidal induces apoptosis and inhibits Akt/mTOR pathway in glioma cells

Background  

The highly resistant nature of glioblastoma multiforme (GBM) to chemotherapy prompted us to evaluate the efficacy of bicyclic triterpenoid Iripallidal against GBM in vitro.     

粉防己碱诱导人视网膜母细胞瘤细胞凋亡及其机制研究

背景与目的：粉防己碱(tetrandrine,Tet)是一种天然化合物,其抗视网膜母细胞瘤作用尚不清楚。该研究拟检测Tet对人视网膜母细胞瘤细胞的抗肿瘤作用,并进一步阐明其作用机制。方法：采用CCK-8法检测Tet对视网膜母细胞瘤细胞活力的抑制作用;应用Annexin V/PI法检测细胞凋亡情况;在2’,7’-二氯荧光素二乙酸酯(2’, 7’-dichlorofluorescin diacetate,DCFH-DA)染色后,采用流式细胞术检测细胞内反应性活性氧(reactive oxygen species,ROS)含量;采用蛋白［质］印迹法(Western blot)检测细胞Akt、p-Akt蛋白表达量。结果：Tet显著抑制视网膜母细胞瘤细胞活力,Tet浓度为4、8、10和20 μmol/L处理细胞24 h时,WERI-Rb-1细胞抑制率分别为5.7%、25.0%、55.1%和84.9%,Y79细胞抑制率分别为2.4%、2.9%、23.8%和54.2% (P<0.01);10 μmol/L Tet处理细胞12、24和48 h时,WERI-Rb-1细胞抑制率分别为6.0%、45.5%和74.7%,Y79细胞抑制率分别为2.9%、19.4%和43.3%(P<0.01)。Tet诱导细胞凋亡,以10 μmol/L Tet处理细胞24和48 h时,WERI-Rb-1细胞凋亡率分别为(23.70±1.75)%和(34.83±3.15)%,Y79细胞凋亡率分别为(9.62±2.69)%和(14.97±1.50)%(P<0.01),凋亡抑制剂Z-VAD-FMK能够显著抑制Tet对视网膜母细胞瘤细胞的凋亡诱导作用(P<0.05)。10 μmol/L Tet作用细胞6及12 h后,细胞的ROS产生量较对照组明显上升(P<0.01),N-乙酰基-L-半胱氨酸(N-acetyl-L-cysteine,NAC)能够抑制Tet诱导产生的ROS(P<0.01),NAC抑制ROS后,细胞的凋亡率较单独Tet作用组明显下降(P<0.01)。Tet能够抑制视网膜母细胞瘤细胞PI3K/Akt信号通路。结论：Tet诱导视网膜母细胞瘤细胞凋亡,该作用机制与细胞内ROS升高、PI3K/Akt信号通路抑制有关。