The anticancer effect of saffron in two p53 isogenic colorectal cancer cell lines

ABSTRACT: BACKGROUND: Saffron extract, a natural product, has been shown to induce apoptosis in several tumor cell lines. Nevertheless, the p53-dependency of saffron's mechanism of action in colon cancer remains unexplored. METHODS: In order to examine saffron's anti-proliferative and pro-apoptotic effects in colorectal cancer cells, we treated two p53 isogenic HCT116 cell lines (HCT wildtype and HCT p53-/-) with different doses of the drug and analyzed cell proliferation and apoptosis in a time-dependent manner. MTT viability and crystal violet assays were performed in order to determine the effective dose of saffron on both cell lines. The cell cycle progress was examined by Flow cytometric analysis. Apoptosis was assessed using Annexin-PI-staining and Western Blotting for caspase 3 and PARP cleavage. Autophagy was determined by Western Blotting of the light chain 3 (LC3)-II and Beclin 1 proteins. The protein content of phospho-H2AX (gammaH2AX), a sensor of DNA double strand breaks, was also analyzed by Western Blotting. RESULTS: Saffron extract induced a p53-dependent pattern of cell cycle distribution with a full G2/M stop in HCT116 p53 wildtype cells. However, it induced a remarkable delay in S/G2 phase transit with entry into mitosis in HCT116 p53 -/- cells. The apoptotic Pre-G1 cell fraction as well as Annexin V staining and caspase 3 cleavage showed a more pronounced apoptosis induction in HCT116 p53 wildtype cells. Obviously, the significantly higher DNA-damage, reflected by H2AX protein levels in cells lacking p53, was coped by up-regulation of autophagy. The saffron-induced LC3-II protein level was a remarkable indication of the accumulation of autophagosomes, a response to the cellular stress condition of drug treatment. Conclusions: This is the first study showing the effect of saffron in HCT116 colorectal cancer cells with different p53 status. Saffron induced DNA-damage and apoptosis in both cell lines. However, autophagy has delayed the induction of apoptosis in HCT116 p53 -/- cells. Considering the fact that most tumors show a functional p53 inactivation, further research is needed to elucidate the long-term effects of saffron in p53 -/- tumors.     

Curcumin induces apoptosis in HCT-116 human colon cancer cells in a p21-independent manner

Several micronutrients present in fruits and vegetables exhibit anticancer activity as a result of their actions on molecular targets involved in carcinogenesis and tumor progression. Curcumin, a phenolic phytochemical derived from the rhizome of Curcuma longa, exhibits both cancer-preventative activity and growth inhibitory effects on neoplastic cells. Several studies report that curcumin inhibits cancer cell proliferation and induces apoptosis in cancer cells through p21-mediated cell cycle arrest. Cancer cells that are deficient in p21 are also reported to be more prone to undergo apoptosis in response to a variety of cytotoxic agents. In this study, we determined whether curcumin-induced cytotoxicity in cultures of HCT-116 human colon cancer cells was dependent on p21 status. Curcumin killed wild-type HCT-116 cells in a dose- and time-dependent manner, as measured in an MTT cell viability assay. Moreover, an equivalent cytotoxic effect by curcumin was observed in both p21^+/+ and p21^−/−HCT-116 cells, indicating that curcumin-induced cytotoxicity was p21-independent. Primary cultures of human dermal fibroblasts were less sensitive than HCT-116 colon cancer cells to lower doses of curcumin, suggesting a degree of selectivity for neoplastic cells. Western blot analysis showed that cell death in curcumin-treated cultures of p21^+/+ and p21^−/− HCT-116 cells was associated with a reduction in pro-caspase-3 and PARP-1 cleavage, which are indicative of apoptosis. We conclude that curcumin-induced apoptosis in HCT-116 colon cancer cells does not depend on p21 status.     

The silence of p66Shc in HCT8 cells inhibits the viability via PI3K/AKT/Mdm-2/p53 signaling pathway

Colon cancer is the second most common cause of cancer-related death, indicating that some of its cancer cells are not eradicated by current therapies. The previous studies demonstrated that p66^Shc protein, a member of Shc family, is highly expressed in colon cancer cells, but the role of p66^Shc in the progress of colon cancer still unknown. In this study, we found that p66^Shc highly expressed in colon cancer tissue and colon cancer cell line SW620 cells, HCT8 cells, HCT116 cells and CaCO₂ cells. The silence of p66^Shc in HCT8 cells reduced the proliferation and accelerated the apoptosis, in addition, the expression of pro-apoptotic proteins caspase-3, caspase-9, Bax was enhanced and the expression of anti-apoptotic protein Bcl-2 was declined. Moreover, the cell cycle arrest in G0/G1 phase after HCT8 cells treated with p66^Shc siRNA. Furthermore, after HCT8 cells treated with p66^Shc siRNA, the phosphorylation of PI3K and AKT was significantly suppressed, and the expression of Mdm-2, a downstream of AKT, was obviously prohibited, while the expression of p53 was enhanced. These results indicate that the silence of p66^Shc in HCT8 cells inhibits the viability via PI3K/AKT/Mdm-2/p53 signaling pathway, it may provide a promising approach to prevent the progress of colon cancer cell.     

DNA methyltransferase 1 is essential for initiation of the colon cancers

DNA methyltransferase 1 (Dnmt1) is essential for the maintenance of hematopoietic and somatic stem cells in mice; however, its roles in human cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are still elusive. In the present study, we investigated DNMT1 functions in the maintenance of human colon CSCs/CICs using the human colon cancer cell line HCT116 (HCT116 w/t) and its DNMT1 knockout cell line (DNMT1^−/−). The rates of CSCs/CICs were evaluated by side population (SP) analysis, ALDEFLUOR assay and expression of CD44 and CD24. SP, ALDEFLUOR-positive (ALDEFLUOR⁺) and CD44-positive and CD24-positive (CD44⁺CD24⁺) cell rates were lower in DNMT1^−/− cells than in HCT116 w/t cells. Since CSCs/CICs have higher tumor-initiating ability than that of non-CSCs/CICs, the tumor-initiating abilities were addressed by injecting immune deficient (NOD/SCID) mice. DNMT1^−/− cells showed less tumor-initiating ability than did HCT116 w/t cells, whereas the growing rate of DNMT1^−/− cells showed no significant difference from that of HCT116 cells both in vitro and in vivo. Similar results were obtained for cells in which DNMT1 had been transiently knocked-down using gene-specific siRNAs. Taken together, these results indicate that DNMT1 is essential for maintenance of colon CSCs/CICs and that short-term suppression of DNMT1 might be sufficient to disrupt CSCs/CICs.     

IFN-gamma induction of p21(WAF1) is required for cell cycle inhibition and suppression of apoptosis.

Interferons (IFN) inhibit the growth of tumor cells by blocking the progression of their cell cycle. Recently, we showed that this cell cycle inhibition correlates with the ability of IFN to upregulate the cyclin-dependent kinase inhibitor p21(WAF1). This, however, is not proof of a causal relationship. Using p21(WAF1)-deficient cells derived from the HCT116 colon adenocarcinoma cell line, we now show that p21(WAF1) is indeed responsible for the antiproliferative effects of the type II IFN, IFN-gamma. IFN-gamma upregulated p21(WAF1) expression in a p53-independent manner, decreased cyclin-dependent kinase 2 activity, and inhibited entry into the S phase of the cell cycle in p21+/+ but not in p21-/- HCT116 cells. We additionally found that the lack of p21(WAF1) expression resulted in an increase in the ability of IFN-gamma to induce apoptosis, as reflected by an earlier induction of DNA fragmentation and caspase 3 activity in p21-/- cell. Our results indicate that p21(WAF1) expression is necessary for IFN-gamma-mediated cell cycle inhibition and suppression of IFN-gamma-induced apoptosis.     

糖皮质激素激活自噬对软骨细胞衰老的影响

目的:探讨糖皮质激素对关节软骨细胞衰老和自噬影响,并分析自噬在其中的作用及机制。方法:培养SD大鼠软骨细胞,阿利辛染色和collagen II免疫组化进行鉴定。使用不同浓度的地塞米松作用于软骨细胞不同的时间后,Lyso Tracker Red和MDC染色观察自噬囊泡的生成,Western blot检测LC3、beclin-1、P62、p70S6K和4EBP1蛋白表达的变化。β-半乳糖苷酶染色分析糖皮质激素对软骨细胞衰老的影响,并使用自噬抑制剂来探讨自噬对地塞米松作用下软骨细胞衰老的作用。结果:地塞米松可以显著提高软骨细胞的自噬水平,尤其是在作用4 d后,LC3-II表达随着地塞米松浓度的增加而升高,而P62和beclin-1的表达也验证自噬水平的升高。Lyso Tracker Red和MDC染色发现软骨细胞中自噬泡的生成随着地塞米松浓度的升高而增加。m TOR信号通路上的p70S6K和4EBP1的磷酸化均被地塞米松抑制。更重要的是,地塞米松可以引起软骨细胞的衰老,而使用3-MA抑制自噬后,细胞进一步衰老。结论:糖皮质激素激活软骨细胞中的自噬,并同时诱导软骨细胞的衰老,而自噬在这过程中对细胞的衰老可能具有代偿性的保护作用。同时糖皮质激素抑制m TOR信号通路,可能与自噬的激活相关。     

Systematic genetic dissection of p14ARF-mediated mitochondrial cell death signaling reveals a key role for p21CDKN1 and the BH3-only protein Puma/bbc3

Induction of cell death by p14^ARF is mediated through a Bax/Bak-dependent mitochondrial apoptosis pathway. To investigate the upstream signaling events required for the activation of Bax and/or Bak and to determine the functional impact of de-regulated cell cycle restriction point control in this context, we genetically dissected the impact of BH3-only proteins and the role of the cyclin-dependent kinase (cdk) inhibitor p21^CDKN1. Using isogenic HCT116 colorectal cancer cells, either wild-type or homozygously deleted for the BH3-only protein Puma/bbc3 and/or p21^CDKN1 or p53-reconstituted DU145 prostate cancer cells, we show that p14^ARF-induced apoptosis is attenuated in the absence of Puma. Upon expression of p14^ARF in HCT116 cells, Puma is rapidly induced at both the mRNA and protein level. Puma-proficient HCT116 cells undergo apoptotic (nuclear) DNA fragmentation, which is preceded by the N-terminal conformational change of Bax, the breakdown of the mitochondrial membrane potential, and induction of caspase-9 (LEHD)-like and caspase-3/7 (DEVD)-like activities. In contrast, p14^ARF-induced apoptosis is markedly attenuated in isogenic HCT116 cells bi-allelically deleted for puma. The sensitivity of Puma-deficient cells to p14^ARF-induced apoptosis is fully restored by functional reconstitution of Puma using a conditional adenoviral expression vector. Notably, the concomitant deletion of p21^CDKN1 strongly enhances p14^ARF-induced apoptosis in Puma-proficient cells, but not in isogenic Puma-deficient cells. These results indicate that p14^ARF-induced mitochondrial apoptosis critically depends on the BH3-only protein Puma. In the presence of a functional p53/Puma/Bax-signaling axis, p14^ARF-triggered apoptosis is enhanced by loss of p21^CDKN1-mediated cell cycle checkpoint control.     

Induction of senescence with doxorubicin leads to increased genomic instability of HCT116 cells

Induction of senescence has been proposed as a possible in vivo tumor response to anticancer treatment. Senescent cancer cells are often polyploid, however, their route to polyploidy is poorly recognized (endoreduplication versus aberrant mitoses). We showed that after treatment of HCT116 cells with a low dose of doxorubicin most of them stopped proliferation as documented by SA-beta-galactosidase activity and the lack of Ki67 expression. Increased expression of other common senescence markers, p53, p21 and cyclin D1, was also observed. The cells became giant, polyploid and polymorphic, with multinucleated cells comprising a substantial fraction. The vast majority of the doxorubicin-treated cells did not enter mitoses, as evidenced by mitotic index analysis, as well as by the predominantly cytoplasmic localization of cyclin B1 and a lack of separation of multiplied centrosomes. This allowed us to conclude that doxorubicin-treated HCT116 cells underwent endoreduplication. However, the rare events of aberrant mitoses of polyploid cells observed by us led to aneuploid progeny as was documented by cytogenetic analysis of survivors. Thus, a senescence-inducing treatment of HCT116 cancer cells had a dual effect-it stopped the proliferation of the majority of the cells, but also led to the appearance of proliferating aneuploid ones.     

CD147对前列腺癌PC-3细胞自噬的影响

 目的：研究白细胞分化抗原（CD）147在体外对前列腺癌PC-3细胞的自噬作用。方法：通过氨基酸饥饿法建立自噬模型,免疫印迹技术检测CD147的表达。利用RNA干扰CD147表达的细胞系,免疫印迹技术检测自噬蛋白LC3-I和LC3-II的表达;台盼蓝排斥实验检测细胞的死亡情况。结果：在PC-3细胞自噬模型中,随着饥饿诱导时间延长,CD147表达逐渐升高。用RNA干扰技术降低CD147表达后,与阴性对照组比较,在自噬模型中CD147干扰组自噬相关蛋白LC3-II表达增多;并且细胞死亡数量明显增加,阴性对照组细胞死亡率分别为（19.3±3.1）%和（22.3±3.5）%,而在CD147干涉组细胞死亡率为(38.4±3.1)%,差异有统计学意义（P<0.05）。结论：在前列腺癌PC-3细胞中CD147抑制饥饿诱导的自噬,减少自噬性细胞死亡的发生。     

Fentanyl inhibits proliferation and invasion of colorectal cancer via β-catenin

Background and aim: Fentanyl is widely used for relieving pain and narcotizing in cancer patients. However, there are few published reports regarding the effects of fentanyl on tumor control and treatment. Here we investigated the effects of fentanyl on tumor growth and cell invasion in the human colorectal carcinoma (HCT116) cells. Methods: Nude mice xenografts of HCT116 cells were established to assess the inhibition effect on tumor growth by fentanyl. MTT and Transwell were employed to determine the cell survival rate and cell invasion, respectively. MicroRNAs and mRNAs expression were quantified by real-time PCR. β-catenin and matrix metalloproteinases (MMP-2 and MMP-9) expression were assayed by western blotting. β-Catenin-specific small interfering RNA (Si-β-catenin) and miR-182 mimics were transfected in cells to investigate the mechanism underlying the effects of fentanyl on the colorectal tumor and HCT116 cells. Results: Treatment with fentanyl inhibited the tumor growth and HCT116 cells invasion. Fentanyl also downregulated the expression of β-catenin and miR-182 in both xenograft tumors and HCT116 cells, and decreased the protein level of MMP-9 in HCT116 cells. Downregulation of β-Catenin resulted in the decrease of miR-182 expression in colorectal cells. In addition, the overexpression of miR-182 reversed the effect of fentanyl on MMP-9 expression and cell invasion of HCT116 cells. Conclusions: The current study demonstrated that the inhibition of tumor growth and cell invasion in colorectal cancer by fentanyl is probably due to downregulation of miR-182 and MMP-9 expression by β-catenin.     

p300/p53/Smad3通路参与人心房成纤维细胞衰老相关心房纤维化

目的:探究p300在人心房成纤维细胞(HAFs)衰老相关心房纤维化中的作用及其可能机制.方法:采用组织贴块法从人左心耳组织中分离HAFs并进行传代培养建立细胞衰老模型,检测衰老相关β-半乳糖苷酶(SA-β-Gal)活性以比较P3和P7代细胞衰老程度的不同.Western blot实验比较不同代数(P3、P7和P11)细...     

Immune recognition of cyclin B1 as a tumor antigen is a result of its overexpression in human tumors that is caused by non-functional p53

Cyclin B1, which plays a key role in the control of cell cycle progression from G(2) through M phase, was recently identified by us as a tumor antigen recognized by human T-cells. To understand what makes this normal molecule antigenic, we compared its expression in malignant versus normal cells. Immunohistology showed overexpression of cyclin B1 protein in tumors compared to surrounding normal tissue and localization in the cytoplasm rather than the nucleus. Cyclin B1 is overexpressed at protein and mRNA level in many tumor cell lines including breast, lung, colorectal carcinoma, lymphoma and leukemia. While overexpressed in tumor cells at all stages of the cell cycle, its expression still peaks at G(2)/M phase, as it does in normal cells. We compared cyclin B1 expression in two cell clones derived from the same colorectal tumor cell line, one wild type for p53 (HCT116p53(+/+)) and one with deleted p53 (HCT116p53(-/-)). HCT116p53(+/+) cells had undetectable (normal) level of cyclin B1 protein, while HCT116p53(-/-) cells showed overexpression. When reconstituted with p53, HCT116p53(-/-) cells reverted to normal cyclin B1 expression. We conclude that p53 plays an important role in cyclin B1 regulation and that tumors with mutated p53 will be good candidates for cyclin B1 based immunotherapy.     

Circulating complexes of the vitamin D binding protein with G-actin induce lung inflammation by targeting endothelial cells

This study investigated the actin scavenger function of the vitamin D binding protein (DBP) in vivo using DBP null (−/−) mice. Intravenous injection of G-actin into wild-type (DBP+/+) and DBP−/− mice showed that contrary to expectations, DBP+/+ mice developed more severe acute lung inflammation. Inflammation was restricted to the lung and pathological changes were clearly evident at 1.5 and 4 h post-injection but were largely resolved by 24 h. Histology of DBP+/+ lungs revealed noticeably more vascular leakage, hemorrhage and thickening of the alveolar wall. Flow cytometry analysis of whole lung homogenates showed significantly increased neutrophil infiltration into DBP+/+ mouse lungs at 1.5 and 4 h. Increased amounts of protein and leukocytes were also noted in bronchoalveolar lavage fluid from DBP+/+ mice 4 h after actin injection. In vitro, purified DBP-actin complexes did not activate complement or neutrophils but induced injury and death of cultured human lung microvascular endothelial cells (HLMVEC) and human umbilical vein endothelial cells (HUVEC). Cells treated with DBP-actin showed a significant reduction in viability at 4 h, this effect was reversible if cells were cultured in fresh media for another 24 h. However, a 24-h treatment with DBP-actin complexes showed a significant increase in cell death (95% for HLMVEC, 45% for HUVEC). The mechanism of endothelial cell death was via both caspase-3 dependent (HUVEC) and independent (HLMVEC) pathways. These results demonstrate that elevated levels and/or prolonged exposure to DBP-actin complexes may induce endothelial cell injury and death, particularly in the lung microvasculature.     

结肠癌细胞HCT116中p53的R213突变对靶基因p21表达的影响

目的确定在结肠癌细胞系中,p53的213位精氨酸突变后对其下游靶基因p21表达的影响。方法在p53缺失的结肠癌细胞系HCT116~(-/-)中转染p53的R213位突变的质粒;荧光素酶双报告基因系统检测p53靶基因启动子活性;EMSA检测p53与其靶基因p21的结合能力;real-time PCR和Western blot检测p53下游靶基因p21的表达。结果 p53蛋白213位精氨酸突变为赖氨酸后,明显降低了p53靶基因的启动子活性(P0.05);与靶基因p21启动子的结合能力明显降低(P0.05);并抑制了p21基因的表达(P0.05)。而213位突变为谷氨酰胺后,迁移率比突变前明显变快。结论在人的结肠癌细胞中,p53蛋白R213位的不同突变对下游靶基因p21有不同的影响,提示存在着不同的调控机制。     

Inflammatory level under different p53 mutation status and the regulation role of curcumin in tumor microenvironment

The inflammation is tightly associated with tumor development, promoting or inhibiting tumorigenesis. And mutant p53 is speculated to promote inflammation and tumorigenesis. The tumor associated macrophages are usually educated to present the anti-inflammatory profile to tune down antitumor immunity. However, the impact of p53 mutants on macrophages is not clear. Here, we compared the basal inflammatory level and macrophage profiles in tumor cells and tumor samples with different p53 mutations. Data revealed that a lower inflammatory level was maintained in immune organs and tumor cells with p53 point mutations than those with p53 null mutation. Using the tumor cell-derived conditional media to culture macrophages, we found that the media from cells with p53 mutations, especially the point mutations, could decrease M1 markers and inhibit phagocytosis, suggesting the p53 mutation promoted M2 profile polarization.To target the p53 mutation induced M2 macrophage polarization, we applied low-concentration curcumin to the tumor cells with different p53 mutations. The data showed that curcumin could inhibit STAT3 signal and decrease PPARγ and CSF1 in tumor cells and tumor samples. In vitro, the co-culture assays showed that the curcumin treatment shifted p53 mutation educated macrophages back towards M1 profile. In vivo, the curcumin-treated MEFs showed obvious tumor inhibition, and the tumor samples displayed inhibited M2 markers. Results suggested that curcumin could inhibit p53 mutation educated macrophage induction and suppress M2-promoted tumorigenesis.Our study illustrated the inflammatory level under different p53 status and the inflammatory regulated role of curcumin in tumor environment. This study might provide a potential method in tumor personalized treatment aiming immune therapy in different p53 status.     

雷公藤甲素通过诱导细胞自噬促进结肠癌CT26细胞死亡

目的探讨雷公藤甲素诱导结肠癌CT26细胞自噬与细胞死亡之间的关系。方法体外培养小鼠结肠癌CT26细胞,应用免疫荧光、荧光双标自噬腺病毒载体(Ad-mRFP-GFP-LC3)技术,在激光扫描共焦显微镜下观察LC3的表达轮廓及水平,并结合免疫印迹法对LC3蛋白、P62蛋白做半定量分析;进而用单溶液细胞增殖检测(MTS)试剂测定CT26细胞增殖抑制率;用Annexin V-FITC/PI双染、流式细胞术检测细胞凋亡/死亡率。结果雷公藤甲素能诱导CT26细胞出现明确的自噬流;激活细胞自噬可提高CT26细胞的晚期凋亡率。与雷公藤甲素组比较,雷帕霉素与其联用能显著增强CT26细胞死亡。结论自噬可能介导了雷公藤甲素诱导CT26细胞死亡的过程。     

Curcumin, but not Prima-1, decreased tumor cell proliferation in the syngeneic murine orthotopic bladder tumor model

OBJECTIVE:

Cigarette smoking is the main risk factor for bladder cancer development. Among the mediators of this effect of smoking is nuclear factor-kappa B. Curcumin suppresses cellular transformation by downregulating the activity of nuclear factor-kappa B. Prima-1 is a compound that induces apoptosis in human tumor cells, restoring the function of mutant p53. Our study aimed to evaluate the effects of curcumin and prima-1 in an animal model of bladder cancer.

METHODS:

Tumor implantation was achieved in six- to eight-week-old female C57BL/6 mice by introducing MB49 bladder cancer cells into the bladder. Intravesical treatment with curcumin and Prima-1 was performed on days 2, 6, 10, and 14. On day 15, the animals were sacrificed. Immunohistochemistry was used to determine the expression of cyclin D1, Cox-2, and p21. Cell proliferation was examined using PCNA.

RESULTS:

Animals treated with curcumin exhibited a higher degree of necrosis than animals in other groups. Immunohistochemistry showed reduced expression of cyclin D1 in the curcumin-treated group. All of the cells in mice treated with curcumin were p21 positive, suggesting that the p53 pathway is induced by this compound. Prima-1 did not induce any change in tumor size, necrosis, cell proliferation, or the expression of proteins related to the p53 pathway in this animal model.

CONCLUSION:

Curcumin showed activity in this animal bladder cancer model and probably acted via the regulation of nuclear factor-kappa B and p53. Therefore, curcumin is a good choice for the use in clinical trials to treat superficial bladder cancer as an alternative to bacillus Calmette-Guerin. In contrast, Prima-1 does not seem to have an effect on bladder cancer.     

From autophagy to senescence and apoptosis in Angiotensin II‐treated vascular endothelial cells

The aim of this study was to explore if cell autophagy is activated by AngII before aging using human umbilical vascular endothelial cells (HUVECs). The ultrastructural analysis of HUVECs was performed to observe autophagosomes. The LC3‐II/LC3‐I ratio was determined by western blot assay. The β‐gal staining was used to identify cell senescence. The flow cytometry was performed to evaluate cell apoptosis. The BH3 domain analog ABT737 or Beclin‐1 knockdown by specific shRNA or valsartan was applied to investigate their effects on cell autophagy, senescence, and apoptosis induced by Ang II. Cell autophagy was initiated after Ang II treatment at 24 h. And cell senescence and apoptosis were observed in Ang II‐treated cells at 48 h. The significant interaction of Beclin‐1 and Bcl‐2 was detected at 48 h after Ang II treatment. Beclin‐1 was indispensable to Ang II‐induced autophagy, and its BH3 domain was required for the interaction with Bcl‐2 to attenuate autophagy. Pretreated with valsartan, cells were present with less autophagic, senescent, and apoptotic cells after Ang II stimulation. In conclusion, Ang II induced autophagy, senescence, and apoptosis of HUVECs progressively, and autophagy presented an early protective effect on vascular endothelial damage due to Ang II.     

Oxidized low-density lipoprotein (oxLDL) induces cell death in neuroblastoma and survival autophagy in schwannoma cells

Oxidized low-density lipoprotein (oxLDL) induces apoptosis or autophagy in dependence on the cell type. We here investigated the effect of oxLDL on the B104 neuroblastoma and RN22 schwannoma cells being popular in neuroscience research. Cells were cultivated with and without oxLDL. To generate oxLDL, we added 50 μg/ml nLDL and 50 μM CuSO₄ into the culture medium. After a 24-h-long treatment, oxLDL was detectable in media from both cell culture types and its concentration was approximately 16 μg/ml. In the oxLDL-treated B104 neuroblastoma cell cultures 75% cells died after the 24-h exposure. The intact cells showed impaired mitochondria at the ultrastructural level. Western blot analysis revealed the increased expression of AIF 57 kDa (AIF₅₇) protein, as a sign of caspase-independent cell death. In RN22 schwannoma cell cultures, oxLDL did not have any effect on cleaved caspase-3 and AIF₅₇ protein levels indicating absence of cell death. Treated RN22 schwannoma cells underwent survival autophagy by forming conspicuous autophagosomes and by processing LC3-I into LC3-II protein.Collectively, oxLDL induces AIF-dependent cell death in B104 neuroblastoma cells whereas in RN22 schwannoma cells enhanced signs of survival autophagy are noted.     

Autophagy inhibition enhances etoposide-induced cell death in human hepatoma G2 cells

Induction of autophagy usually acts as a survival mechanism of cancer cells in response to chemotherapy. However, the function and molecular mechanism of autophagy in human hepatoma cells under drug treatment is still not clear. To address this issue, we established an experimental model in which HepG2 cells were treated with etoposide, a widely used anticancer agent. We demonstrate the etoposide-induced accumulation of GFP-LC3 dots by fluorescent microscopy, the up-regulation of LC3-II protein expression by Western blotting and the increased number of autophagic vacuoles by electron microscopy, confirming the activation of autophagy by etoposide in HepG2 cells. Inhibition of autophagy by either 3-methyladenine (3MA) or beclin-1 small interfering RNA enhanced etoposide-induced cell death. Furthermore, activation of p53 and AMPK was detected in etoposide-treated cells and inhibition of AMPK triggered apoptosis through suppression of autophagy. On the other hand, inactivation of p53 promoted cell survival through augmentation of autophagy. Collectively, these findings indicate that etoposide-induced autophagy promotes hepatoma cell adaptation and survival, and that autophagy inhibition improves the chemotherapeutic effect of etoposide. Moreover, AMPK activation is clearly associated with etoposide-induced autophagy. We conclude that manipulation of AMPK may be a promising approach of adjuvant chemotherapy for hepatocellular carcinoma.