Nakagawa-Goto K, Chen JY, Cheng YT, Lee WL, Takeya M, Saito Y, Lee KH, Shyur LF

Nakagawa-Goto K, Chen JY, Cheng YT, Lee WL, Takeya M, Saito Y, Lee KH, Shyur LF. (within 2 h treatment) and damage to the ER structures, resulting in ER-derived cytoplasmic vacuolation and ubiquitinated protein accumulation in the treated cells. Intriguingly, the effects of both compounds were blockaded by pretreatment with ROS scavengers, L. or other genus plants. In Chinese medicine, is used for treating hepatitis, bronchitis, nephritis, arthralgia, or stomach disease symptoms [6, 7]. In our previous studies, we observed that DET is the active compound in the medicinal plant Arecoline which was found to significantly suppress mammary tumor growth and lung metastasis of TS/A (ER+) mammary cancer cells and effect of both compounds against MDA-MB-231 cell activity in an orthotopic tumor model using NOD/SCID mice [11]. We observed that treatment with DETD-35 (10 mg/kg/every three days, < 0.05) (Supplementary Figure 1). The and data demonstrate that DETD-35 has a more potent effect than the parental DET against triple unfavorable breast malignancy cell proliferation and growth. Open in a separate window Physique 1 Effects of DET and DETD-35 on MDA-MB-231 cells(A) Chemical structure of paclitaxel (PTX), deoxyelephantopin (DET) and its derivative DETD-35; MDA-MB-231 and MCF-10A cells were treated with the indicated concentrations of DET, DETD-35, and PTX for 24 h, and then the cell viability was examined using MTT assay. (B) MDA-MB-231 cells were treated with vehicle (0.5% DMSO), DET (11 M), DETD-35 (3 M), and PTX (1 M) for 24 h, and the morphological changes of cancer cells were examined by light microscopy (400 magnification). (C) Transmission electron microscopy (TEM) imaging (10,000 magnification) of untreated (vehicle) and treated (DET, 11 M; DETD-35, 3 M; PTX, 1 M) MDA-MB-231 cells. The ER and mitochondria (mt) are indicated by black arrowheads and white arrowheads, respectively. Further, both DET and DETD-35 at 11 M and 3 M, respectively, significantly induced the formation of massive cytoplasmic vacuoles in the perinuclear region of MDA-MB-231 cells treated for 24 h, as examined by light microscopy. PTX treatment (1 M) also generated some vacuole-like structures near the nuclear region of MDA-MB-231 cells (Physique ?(Figure1B).1B). We further examined the detailed morphology of treated TNBC cells using transmission electron microscopy (TEM). As shown in Figure ?Physique1C,1C, after treatment for 24 h, several Arecoline vacant vacuoles had appeared in DET- and DETD-35-treated MDA-MB-231 cells with the plasma membrane retained intact, but with a lack of detectable cytoplasmic materials. PTX treatment induced the appearance of multiple micronuclei within cells, and generated several vacuole-like structures made up of rich and dense contents; different from the observations for DET or Arecoline DETD-35 treatment (Physique ?(Physique1C).1C). The multiple ribosomes embedded on the rough endoplasmic reticulum (RER) membrane, a feature of RER structures, were found in the vehicle and PTX-treated TNBC cells, but Arecoline were not seen after either DET or DETD-35 treatment. Meanwhile, both DET and DETD-35 caused significant damage to the mitochondrial structures in the treated TNBC cells. A large populace of swollen mitochondria was observed in DETD-35-treated cells and severe damage to mitochondria structural integrity was observed in DET-treated cells in comparison to vehicle-treated cells. PTX treatment did not cause any apparent mitochondrial damage, except obvious multi-nuclei formation. Together, these results indicate that both DET and DETD-35 treatment induced the formation of massive cytoplasmic vacuoles and damaged the integrity of ER and mitochondrial structures in Rabbit polyclonal to ADNP human TNBC cells; and the effect seen was obviously different from the PTX effect. DETD-35 promotes non-autophagic cytoplasmic vacuolation death in TNBC cells To further pinpoint the potential molecular mechanisms of DET- and DETD-35-induced cytoplasmic vacuolation in inhibition of TNBC cell activity, we first examined whether compound-stimulated cytoplasmic vacuolation is related to autophagic cell death. The accumulation of autophagic vacuoles has been reported to promote cancer cell death through deregulation of lysosomal membrane permeabilization [16]. In addition, the different stages of the autophagic process can be indicated using distinct autophagy marker proteins. We, therefore, used two different kinds of.