Methyl-β-cyclodextrin potentiates the BITC-induced anti-cancer effect through modulation of the Akt phosphorylation in human colorectal cancer cells

ABSTRACT

Methyl-β-cyclodextrin (MβCD) is an effective agent for the removal of plasma membrane cholesterol. In this study, we investigated the modulating effects of MβCD on the antiproliferation induced by benzyl isothiocyanate (BITC), an ITC compound mainly derived from papaya seeds. We confirmed that MβCD dose-dependently increased the cholesterol level in the medium, possibly through its removal from the plasma membrane of human colorectal cancer cells. The pretreatment with a non-toxic concentration (2.5 mM) of MβCD significantly enhanced the BITC-induced cytotoxicity and apoptosis induction, which was counteracted by the cholesterol supplementation. Although BITC activated the phosphoinositide 3-kinase (PI3K)/Akt pathway, MβCD dose-dependently inhibited the phosphorylation level of Akt. On the contrary, the treatment of MβCD enhanced the phosphorylation of mitogen activated protein kinases, but did not potentiate their BITC-induced phosphorylation. These results suggested that MβCD might potentiate the BITC-induced anti-cancer by cholesterol depletion and thus inhibition of the PI3K/Akt-dependent survival pathway.

Abbreviations: CDs: cyclodextrins; MβCD: methyl-β-cyclodextrin; ITCs: isothiocyanates; BITC: benzyl isothiocyanate; PI3K: phosphoinositide 3-kinase; PDK1: phosphoinositide-dependent kinase-1; MAPK: mitogen activated protein kinase; ERK1/2: extracellular signal-regulated kinase1/2; JNK: c-Jun N-terminal kinase; PI: propidium iodide; FBS: fatal bovine serum; TLC: thin-layer chromatography; PBS(-): phosphate-buffered saline without calcium and magnesium; MEK: MAPK/ERK kinase; PIP2: phosphatidylinositol-4,5-bisphosphate; PIP3: phosphatidylinositol-3,4,5-trisphosphate     

Bystander CD4 T-cell death is inhibited by broadly neutralizing anti-HIV antibodies only at levels blocking cell-to-cell viral transmission

The progressive loss of CD4+ T cells during HIV infection of lymphoid tissues involves both the apoptotic death of activated and productively infected CD4 T cells and the pyroptotic death of large numbers of resting and abortively infected bystander CD4 T cells. HIV spreads both through cellular release of virions and cell-to-cell transmission involving the formation of virological synapses. Cell-to-cell transmission results in high-level transfer of large quantities of virions to the target cell exceeding that achieved with cell-free virions. Broadly neutralizing anti-HIV antibodies (bNAbs) binding to HIV envelope protein capably block cell-free virus spread, and when added at higher concentrations can also interdict cell-to-cell transmission. Exploiting these distinct dose–response differences, we now show that four different bNAbs block the pyroptotic death of bystander cells, but only when added at concentrations sufficient to block cell-to-cell transmission. These findings further support the conclusion that HIV killing of abortively infected bystander CD4 T cells requires cell-to-cell transfer of virions. As bNAbs attract more interest as potential therapeutics, it will be important to consider the higher concentrations of these antibodies required to block the inflammatory death of bystander CD4 T cells.     

Dieckol,a natural polyphenolic drug,inhibits the proliferation and migration of colon cancer cells by inhibiting PI3K,AKT, and mTOR phosphorylation

This study investigated that dieckol (DKL), a natural drug, inhibits colon cancer cell proliferation and migration by inhibiting phosphoinositide-3-kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) phosphorylation in HCT-116 cells. The cells were treated with DKL in various concentrations (32 and 50 μM) for 24 h and then analyzed for various experiments. MTT (tetrazolium bromide) and crystal violet assay investigated DKL-mediated cytotoxicity. Dichlorodihydrofluorescein diacetate staining was used to assess the reactive oxygen species (ROS) measurement, and apoptotic changes were studied by dual acridine orange and ethidium bromide staining. Protein expression of cell survival, cell cycle, proliferation, and apoptosis protein was evaluated by western blot analysis. Results indicated that DKL produces significant cytotoxicity in HCT-116, and the half-maximal inhibitory concentration was found to be 32 μM for 24-h incubation. Moreover, effective production of ROS and enhanced apoptotic signs were observed upon DKL treatment in HCT-116. DKL induces the expression of phosphorylated PI3K, AKT, and mToR-associated enhanced expression of cyclin-D1, proliferating cell nuclear antigen, cyclin-dependent kinase (CDK)-4, CDK-6, and Bcl-2 in HCT-116. In addition, proapoptotic proteins such as Bax, caspase-9, and caspase-3 were significantly enhanced by DKL treatment in HCT-116. Hence, DKL has been considered a chemotherapeutic drug by impeding the expression of PI3K-, AKT-, and mTOR-mediated inhibition of proliferation and cell cycle-regulating proteins.     

Activation of JNK/c-Jun is required for the proliferation, survival, and angiogenesis induced by EET in pulmonary artery endothelial cells

Pulmonary artery endothelial plexiform lesion is responsible for pulmonary vascular remodeling (PVR), a basic pathological change of pulmonary arterial hypertension (PAH). Recent evidence suggests that epoxyeicosatrienoic acid (EET), which is derived from arachidonic acid by cytochrome p450 (CYP) epoxygenase, has an essential role in PAH. However, until now, most research has focused on pulmonary vasoconstriction; it is unclear whether EET produces mitogenic and angiogenic effects in pulmonary artery endothelial cells (PAEC). Here we found that 500 nM/l 8,9-EET, 11,12-EET, and 14,15-EET markedly augmented JNK and c-Jun activation in PAECs and that the activation of c-Jun was mediated by JNK, but not the ERK or p38 MPAK pathway. Moreover, treatment with 8,9-EET, 11,12-EET, and 14,15-EET promoted cell proliferation and cell-cycle transition from the G0/G1 phase to S phase and stimulated tube formation in vitro. All these effects were reversed after blocking JNK with Sp600125 (a JNK inhibitor) or JNK1/2 siRNA. In addition, the apoptotic process was alleviated by three EET region isomers through the JNK/c-Jun pathway. These observations suggest that 8,9-EET, 11,12-EET, and 14,15-EET stimulate PAEC proliferation and angiogenesis, as well as protect the cells from apoptosis, via the JNK/c-Jun pathway, an important underlying mechanism that may promote PAEC growth and angiogenesis during PAH.     

XIAP及survivin 在原发性肝细胞癌组织中的表达及其相关性研究

目的：探讨X 连锁凋亡抑制蛋白（XIAP）和survivin 在原发性肝细胞癌中的表达及两者的相关性。方法：选取本院收治的60 例原发性肝细胞癌患者,应用免疫组织化学染色的方法对肝癌组织及癌旁组织中的XIAP及survivin 的表达进行检测。结果：经 比较,肝癌组织中XAIP 及survivin 的阳性率均显著高于癌旁组织,差异有统计学意义。XIAP 和survivin 的表达强度与肿瘤的大 小无关,但随肿瘤的分化程度的降低而升高,且不同分化程度之间差异有统计学意义;XIAP 和survivin 存在正相关关系。结论： XIAP与Survivin 在肿瘤组织中的高表达在促进肿瘤发生、增殖、转移以及耐药,并且能够降低肿瘤的分化程度,增加肝癌的恶性 程度。此外,两者可能存在协同作用,但两者的相关性及作用机制仍需进一步探讨。     

JNK相互作用蛋白通过JNK途径影响鼻咽癌的增殖和凋亡

EB病毒编码的瘤蛋白潜伏膜蛋白（LMP1）所介导的活化蛋白（AP-1）信号转导途径在细胞增殖、分化、转化与凋亡方面发挥着重要作用.越来越多的证据表明,AP-1信号转导通路中上游激酶JNK在鼻咽癌的发生发展过程中起着重要作用.最近克隆出来的JNK相互作用蛋白(JIP-1)是一种能抑制JNK核移位的胞浆锚蛋白.为探讨JIP在LMP1调控AP-1信号通路中的作用机制,采用间接免疫荧光法和报告基因法,发现JIP通过有效地抑制磷酸化的JNK从胞浆移位入核,从而抑制LMP1上调的AP-1活性.同时,JIP导入鼻咽癌细胞中,MTT法发现JIP能够明显抑制鼻咽癌细胞的生长.进一步发现转染JIP后细胞的集落形成率与对照组相比大约降低了53.6%,也抑制了细胞. 提示JIP可明显抑制细胞的增殖作用.进一步采用流式细胞术分析,结果发现JIP引起细胞G1/S期细胞阻滞,说明JIP是抑制细胞增殖的重要调节子.进一步采用流式细胞术定量发现,转染JIP后细胞的24 h凋亡百分率由1.25%上升到8.25%,上升约6.6倍,48 h由1.04%上升到31.45%,上升约30倍. 采用激光共聚焦显微镜发现,转染JIP后细胞核发生显著变化,核质由均匀状态固缩成高凝集状态,形成了典型的胞膜体.提示JIP可有效地促进细胞凋亡.结果表明,JIP可通过抑制活化的JNK核移位,降低LMP1所介导的AP-1信号通路.并进一步发现JIP可有效地抑制细胞增殖和细胞凋亡,从而提示JIP可作为新的治疗肿瘤潜在靶分子.     

Hepatic Sinusoidal Endothelium Heterogeneity with Respect to the Recognition of Apoptotic Cells

The recognition and removal of human apoptotic peripheral lymphocytes in selected populations of periportal and perivenous endothelial cells was studied inin situandin vitroexperiments. Apoptotic peripheral blood lymphocytes once injected into the liver circulation were retained by the sinusoids showing a large heterogeneity of distribution: apoptotic cells are found in the periportal tract double the amount found in the perivenous region. Apoptotic PBL adhesion was lowered to a sixth of the control after preinjection with a sugar mixture (Mannose,N-acetylgalactosamine,N-acetylglucosamine,d-galactose), as suggested by the expression of modified surface glycoconjugates on the plasma membrane of apoptotic cells. A bimodal profile of the distribution of the hepatic sinusoidal cell population, regarding the number of galactose and mannose receptors and the porosity index, was found. Two endothelial cell subsets were present: low porosity cells (average index 14 ± 6%; periportal tract) with a high number of carbohydrate binding sites, and high porosity cells (average index 26 ± 7%; perivenous tract), with a low number of carbohydrate binding sites.     

Methylparaben induces malformations and alterations on apoptosis,oxidant–antioxidant status,ccnd1 and myca expressions in zebrafish embryos

Methylparabens (MP) are widely used as preservatives in cosmetics, pharmacy, and food industry. Although acute toxicity studies in animals indicated that parabens are not significantly toxic, the effects of chronic exposure under sublethal doses are still unknown and the number of related studies is limited. Our aim was to evaluate the effects of MP on the development of zebrafish embryos focusing on development, locomotor activity, oxidant–antioxidant status, apoptosis, and ccnd1 and myca expressions. The expressions of ccnd1 and myca were determined by RT‐PCR. Lipid peroxidation (LPO), nitric oxide (NO), and glutathione‐S‐transferase (GST) activities were determined spectrophotometrically. Apoptosis was determined using acridine orange staining. Locomotor activity was measured using touch‐evoked movement test. MP exposure increased malformations, LPO, apoptosis, ccnd1 and myca expressions, and decreased GST activities and NO levels compared with the control group. Our findings will lead to further understanding of the mechanism of MP toxicity, and merit further research.