Smac N7 Peptide Research Articles

Development of injectable thermosensitive polypeptide hydrogel as facile radioisotope and radiosensitizer hotspot for synergistic brachytherapy

Brachytherapy is considered to be an unparalleled form of conformal radiation therapy, which involves the delivery of radiation directly to tumor lesions or the postoperative cavity. With the development of specific applicators, the exploitation of in situ drug-delivery platform introduces opportunities for the synchronous administration of radiosensitizers. In this study, an iodine-131 (I131)-labeled injectable thermosensitive methoxy poly(ethylene glycol)-b-poly(tyrosine) hydrogel (denoted as PETyr-I131) was developed via a facile method. The radioactive source of I131 was immobilized at the subcutaneous injection site and monitored via single-photon emission computed tomography in real time, and hematological and histopathological analyses revealed no obvious side effects. Additionally, the SmacN7 peptide conjugated with cell membrane-permeable oligosarginine (denoted as SmacN7-R9) was used to enhance the radiosensitivity of cancer cells, as confirmed by the results of reactive oxygen species detection, DNA damage assay, cell apoptosis assay, and clonogenic evaluation. Importantly, a synergistic brachytherapy treatment effect on tumor-bearing nude mice was achieved. The proposed thermosensitive supramolecular hydrogel platform, which conformally immobilizes radionuclides and delivers radiosensitizers by virtue of its proximity to the site of the primary tumor or the postoperative cavity, has great potential for achieving synergistic treatment outcomes with reduced radiation-related side effects. Statement of SignificanceIn this work, a kind of radioiodinated thermosensitive supramolecular hydrogel was developed, which was facilely used as the radioactive source for brachytherapy. Meanwhile, SmacN7-R9 peptide was combined as a model radiosensitizer to facilitate the activation of tumor cell apoptosis pathways and promotion of radiation-induced cytotoxicity. Synergistic brachytherapy outcomes were achieved from the in vitro and in vivo evaluations. Therefore, from the practical standpoint, this thermosensitive supramolecular hydrogel platform holds great potential for the 3D-conformally immobilizing radionuclide and delivering radiosensitizer by virtue of its proximity to the site of primary tumor lesions or postoperative cavity, resulting in synergetic treatment outcomes with reduced radiation associated side effects.

Structure-based design, synthesis and anticancer effect of cyclic Smac-polyarginine peptides.

The second mitochondria-derived activator of caspase (Smac/DIABLO) is a pro-apoptotic protein that released from mitochondria into the cytosol when cells undergo apoptosis. Smac promotes caspase activation by binding the inhibitors of apoptosis proteins (IAP), particularly XIAP and eliminating their inhibitory activity. Although the seven N-terminal amino acids AVPIAQK (SmacN7) of Smac protein is able to elicit an anticancer response by itself, it is neither cell-permeable nor stable in the cellular environment. Thus, the use of SmacN7 derivatives and mimetics is an alluring field for cancer therapy. In this study, heptamer Smac peptide was fused to a well-known octaarginine cell-penetrating peptide for promoting its intracellular access. Both therapeutic Smac part and cell-penetrating octaarginine parts of the peptide sequence constrained in a cyclic structure so as to enhance the apoptosis-inducing potential of the SmacN7 peptide. Biological assays interestingly showed that cyclic peptides P4, P5 and P7 gave rise to a significant level of cytotoxicity and apoptosis mediated cell death in multiple myeloma tumor cells (MM) comparing to linear peptide.

Chemosensitization of Prostate Carcinoma Cells with a Receptor-directed Smac Conjugate.

Second mitochondrial activator of caspase (Smac) is a short mitochondrial peptide. When released from the mitochondria into the cytoplasm, it binds to inhibitor of apoptotic proteins (IAPs) within the cytoplasm and prevents them from inhibiting apoptosis. Delivery of external synthetic Smac peptide into the cytoplasm of malignant cells could greatly improve the efficiency of apoptosis-inducing chemotherapeutic agents. In our study different conjugates based on the seven N-terminal amino acids AVPIAQK of Smac (SmacN7) were produced to obtain a cytoplasm-directed Smac variant. SmacN7 and a point mutant (AVPKAQK) were coupled either to rhodamine alone or to both rhodamine and undecylic aldehyde, which is an antagonist of the Lily-of-the-valley fragrance receptor. The fifth conjugate consisted of rhodamine coupled only to undecylic aldehyde, without SmacN7. The uptake of these five conjugates into three different human cell lines was characterized and quantified by confocal laser scanning microscopy and flow cytometry. A caspase apoptosis assay was performed for cells incubated with the five different conjugates after induction of apoptosis. The coupling of undecylic aldehyde to SmacN7 increased the cellular uptake of the correct and mutant conjugates. Caspase 3/7 apoptosis tests after induction of apoptosis with staurosporine or UV irradiation showed that the coupling of SmacN7 with undecylic aldehyde resulted in a greatly increased adjuvant pro-apoptotic effect compared to the separate components and a mutant SmacN7 peptide sequence in the LNCaP prostate carcinoma cells compared to the benign prostate hyperplasia (BPH) cells and the human embryonal kidney (HEK) cells.

Smac therapeutic Peptide nanoparticles inducing apoptosis of cancer cells for combination chemotherapy with Doxorubicin.

Smac-conjugated nanoparticle (Smac-NP) was designed to induce the apoptosis of cancer cells and as a drug carrier for combination therapy. It contained three parts, a SmacN7 peptide which could induce apoptosis of cancer cells by interacting with XIAPs, the cell penetrating domain rich in arginine, and four hydrophobic tails for self-assembled Smac-NP. We demonstrated that Smac-NPs exerted an antitumor effect in breast cancer cell MDA-MB-231 and nonsmall lung cancer (NSCLC) cell H460, which efficiently inhibited cancer cells proliferation without influencing normal liver cell lines LO2. Smac-NPs also significantly induced apoptosis of MDA-MB-231 and H460 cells through activating pro-caspase-3, down-regulating the expression of antiapoptotic protein Bcl-2 and up-regulating the pro-apoptotic protein Bax. Furthermore, Smac-NPs could be explored as a drug delivery system to load hydrophobic drug such as DOX for combination therapy. The DOX-loaded nanoparticles (DOX-Smac-NPs) exhibited higher cellular uptake efficiency and antitumor effect. Our work provided a new insight into therapeutic peptides integrated with drug simultaneously in one system for cancer combination treatment.

Reversal of Chemoresistance in Human Lung Cancer Cell Line A549/Taxol by Synthetic Second Mitochondria-Derived Activator of Caspase Peptide

Chemoresistance is a leading cause of treatment failure in advanced lung cancer, including that with the extensively prescribed taxol. Recently, a series of structurally unique second mitochondria-derived activators of caspase (Smac) that act as antagonists of inhibitor of apoptosis proteins (IAPs) have been discovered, exhibiting the ability of inducing enhanced apoptosis of various cancer cell types when combined with chemotherapy. In the present study, we synthesized the second mitochondria-derived activator of caspase peptide (Smac-N7 for short) and explored its capacity in combination with taxol in vitro. The sensitivity assay and reversal ability of Smac-N7 were tested by MTT. Flow cytometry was used to analyze apoptosis of cells with Annexin V/PI double staining technique. Cell cloning ability was performed to reflect its biological behavior in each group. Concentrations with inhibitory rates < 10% were selected as the reversal value of Smac-N7 peptide using MTT. The reversal folds were 2.52, 3.26, 3.67, and 5.4 in taxol + Smac-N7 (0.0390625, 0.078125, 0.15625, 0.3125 μg/mL, respectively), and concentrations of Smac-N7 and reversal folds appeared in an obvious positive correlation (r(s) = 1, p = 0.000). Apoptosis analyzed at 48 hours by flow cytometry showed the apoptotic rates in taxol and 0.0390625, 0.078125, 0.15625, and 0.3125 μg/mL Smac-N7 + taxol groups were 15.4 ± 1.09%, 20.8% ± 2.18%, 28.4% ± 4.17%, 37.64% ± 6.41%, and 46.6% ± 7.76%, respectively. Concentrations of Smac-N7 appeared to have negative correlations with PE and SF (r(s) = -1, p < 0.05), which showed that the cells' cloning ability in 0.3125 μg/mL Smac-N7 + taxol group was worse than that of other groups. When combined with taxol, 0.3125 μg/mL Smac-N7 peptide may significantly increase taxol-induced apoptosis in chemoresistant A549/taxol lung cells at 48 hours, and is potentially useful as a reversal agent in lung cancer therapy.

Smac peptide potentiates TRAIL- or paclitaxel-mediated ovarian cancer cell death in vitro and in vivo

Second mitochondria-derived activator of caspases (Smac) is a recently identified protein that is released from mitochondria in response to apoptotic stimuli and promotes apoptosis by antagonizing the inhibitor of apoptosis proteins (IAPs). Our previous study showed that ectopic overexpression of Smac sensitizes drug-resistant tumor cells to TRAIL- or paclitaxel-induced apoptosis in vitro. The present study was designed to explore the effect of the synthesized Smac N7 peptide in a human ovarian cancer cell line and xenograft model. The results showed that the single-agent Smac N7 had a non-cytotoxic effect, but it effectively enhanced TRAIL- or paclitaxel-induced inhibition of cell proliferation in a dose-dependent manner, even in TRAIL-resistant A2780 cells. When Smac N7 was combined with TRAIL or paclitaxel in treating A2780 cell tumor xenografts, synergistic anticancer effects were achieved. Furthermore, the combination therapy caused less damage in normal tissues and more apoptosis in tumor xenografts compared with TRAIL or paclitaxel alone. Increased apoptosis was associated with the downregulation of XIAP, survivin and the increased activity of caspase-3, along with an increased amount of cleaved PARP. In conclusion, this Smac N7 peptide is a promising candidate for ovarian cancer combination therapy, and Smac may be the target for the development of a novel class of anticancer drugs.

NOXA-Induced Alterations in the Bax/Smac Axis Enhance Sensitivity of Ovarian Cancer Cells to Cisplatin

Ovarian cancer is the most common cause of death from gynecologic malignancy. Deregulation of p53 and/or p73-associated apoptotic pathways contribute to the platinum-based resistance in ovarian cancer. NOXA, a pro-apoptotic BH3-only protein, is identified as a transcription target of p53 and/or p73. In this study, we found that genetic variants of Bcl-2 proteins exist among cisplatin-sensitive and -resistant ovarian cancer cells, and the responses of NOXA and Bax to cisplatin are regulated mainly by p53. We further evaluated the effect of NOXA on cisplatin. NOXA induced apoptosis and sensitized A2780s and SKOV3 cells to cisplatin in vitro and in vivo. The effects were mediated by elevated Bax expression, enhanced caspase activation, release of Cyt C and Smac into the cytosol. Furthermore, gene silencing of Bax or Smac significantly attenuated NOXA and/or cisplatin-induced apoptosis in chemosensitive A2780s cells, whereas overexpression of Bax or addition of Smac-N7 peptide significantly increased NOXA and/or cisplatin-induced apoptosis in chemoresistant SKOV3 cells. To our knowledge, these data suggest a new mechanism by which NOXA chemosensitized ovarian cancer cells to cisplatin by inducing alterations in the Bax/Smac axis. Taken together, our findings show that NOXA is potentially useful as a chemosensitizer in ovarian cancer therapy.

SMAC-N7 PEPTIDE WAS MORE POTENT THAN XIAP GENE TARGETING IN SENSITIZING RENAL CANCER CELLS TO APOPTOTIC STIMULI

253 SMAC-N7 PEPTIDE WAS MORE POTENT THAN XIAP GENE TARGETING IN SENSITIZING RENAL CANCER CELLS TO APOPTOTIC STIMULI Tomoyuki Kato*, Sei Naito, Hisashi Kawazoe, Tomohiro Shibasaki, Yuko Nakano, Takuya Yamanobe, Akira Kajinuma, Akinori Muto, Bilim Vladimir, Akira Nagaoka, Yoshihiko Tomita. Yamagata, Japan. INTRODUCTION AND OBJECTIVE: Renal cell carcinoma (RCC) is known to be resistant to chemoand radiotherapy due to a high apoptotic threshold. Inducing apoptosis in RCC cells leads to tumor regression and a molecule responsible for avoiding apoptosis can be a biomarker of molecular target therapy. In this study we tried to manipulate function of the strong anti-apoptotic molecule, XIAP, using RNAi and a peptide mimicking its inhibitory molecule, the second mitochondriaderived activator of caspase (Smac), releasing from mitochondria when a cell is exposed to apoptotic stimuli. METHODS: The surgical specimens from 34 patients who underwent surgery for RCC from January to December 2005 at the Yamagata University Hospital were subjected to immunohistochemical staining with anti-Smac and XIAP antibodies. Staining score was calculcuated by staining intensity multiplied by percent of positive cells, range 0-300. Caki1 RCC cell line with high levels of XIAP was transfected with XIAP targeting shRNA vector and stable clones were generated. The cell permeable peptide Smac-N7 was applied to in vitro treatment of the Caki1. Detection of apoptosis was performed by Hoechst 33342 and Giemsa staining and cell viability was checked by MTS assay. RESULTS: Smac was found in all tumors with staining scores varying from 200 to 300 and staining intensity was weaker than in normal kidneys. XIAP expression levels increased from pT1 (mean score 164.4±108.2) to pT2(195.0±83.3) and pT3(266.7±57.7), as well as from grade 1 (75.0± 98.7) to grade 2 (201.2 ± 93.6) (p<0.05). XIAP was knocked down by RNAi in clone #14 by 81.6% and in clone #19 by 85.3%. Compared to the parental and mock-transfected cells, neither clone was more sensitive to conventional chemotherapeutic agents, but both clones were more susceptible to Fas-stimulation (p<0.0001) and to pharmacological Bcl-2 inhibition (p<0.0001), as well as to a combination of the two (p<0.0001). Exposure of Caki1 cells to Smac-N7 peptide

Role of Inhibitor of Apoptosis Proteins and Smac/DIABLO in Staurosporine-induced Cerebellar Granule Neurons Death

Apoptosis is characterized by morphological and biochemical alterations mediated by caspases. The Inhibitor of Apoptosis Proteins (IAP) comprises a family of proteins that regulate apoptosis by inhibiting caspases. IAP are controlled by different mechanisms including transcriptional regulation, ubiquitination and interaction with proapoptotic proteins such as Smac/DIABLO. Here, we evaluated the role of IAP and Smac/DIABLO in neuronal death. We used cultured rat cerebellar granule neurons (CGN) under conditions that induce apoptosis (staurosporine). We found the expression of cIAP-1, cIAP-2, XIAP and survivin, but not BRUCE in CGN under survival conditions. When CGN were treated with staurosporine we detected a decrease in cIAP-1 and cIAP-2, but not in XIAP and survivin levels. Under these conditions Smac/DIABLO was released from the mitochondria suggesting its involvement in staurosporine-induced death of CGN. However, the Smac N7 peptide, which interacts with caspase-9 binding site in XIAP, did not show any effect on CGC viability.

Double inhibition of XIAP and Bcl-2 axis is beneficial for retrieving sensitivity of renal cell cancer to apoptosis

Renal cell carcinoma (RCC) is known to be resistant to chemo- and radiotherapy due to a high apoptotic threshold. Smac and XIAP (X-linked inhibitor of apoptosis protein) proteins were detected in all RCC cell lines and tissue samples examined. We modulated the function of XIAP, either through its constitutional downregulation with an shRNA vector or by applying a Smac-mimicking peptide. Among RCC cell lines, Caki1 expresses the highest levels of XIAP. We transfected Caki1 with XIAP-targeting shRNA vector and generated stable clones. XIAP was knocked down by RNA interference in clone no. 14 by 81.6% and in clone no. 19 by 85.3%. Compared to the parental and mock-transfected cells, neither clone was more sensitive to conventional chemotherapeutic agents, but both clones were more susceptible to Fas stimulation (P<0.0001) and to pharmacological Bcl-2 inhibition (P<0.0001), as well as to a combination of the two (P<0.0001). Mature Smac binds to XIAP via the N-terminal residues, disrupting its interaction with caspases and promoting their activity. We determined that exposure of Caki1 cells to Smac-N7 peptide (AVPIAQK) resulted in a slight but significant decrease in viability (P=0.0031) and potentiated cisplatin's effect (P=0.0027). In contrast with point targeting of XIAP by shRNA, Smac-N7 peptide is active against several IAP (inhibitor of apoptosis protein) family members, which can explain its role in sensitising cells to cisplatin. Our results suggest that multiple targeting of both Bcl-2 and XIAP or, alternatively, of several IAP family members by the Smac-N7 peptide is a potent way to overcome resistance of RCC to apoptosis-triggering treatment modalities, and might be a new tool for molecular targeted therapy.

Sensitization of TRAIL-resistant LNCaP cells by resveratrol (3, 4', 5 tri-hydroxystilbene): molecular mechanisms and therapeutic potential

BackgroundWe have previously shown that prostate cancer LNCaP cells are resistant to TRAIL, and downregulation of PI-3K/Akt pathway by molecular and pharmacological means sensitizes cells to undergo apoptosis by TRAIL and curcumin. The purpose of this study was to examine the molecular mechanisms by which resveratrol sensitized TRAIL-resistant LNCaP cells.ResultsResveratrol inhibited growth and induced apoptosis in androgen-dependent LNCaP cells, but had no effect on normal human prostate epithelial cells. Resveratrol upregulated the expression of Bax, Bak, PUMA, Noxa, Bim, TRAIL-R1/DR4 and TRAIL-R2/DR5, and downregulated the expression of Bcl-2, Bcl-XL, survivin and XIAP. Treatment of LNCaP cells with resveratrol resulted in generation of reactive oxygen species, translocation of Bax and p53 to mitochondria, subsequent drop in mitochondrial membrane potential, release of mitochondrial proteins (cytochrome c, AIF, Smac/DIABLO and Omi/HtrA2), activation of caspase-3 and caspase-9 and induction of apoptosis. The ability of resveratrol to sensitize TRAIL-resistant LNCaP cells was inhibited by dominant negative FADD, caspase-8 siRNA or N-acetyl cysteine. Smac siRNA inhibited resveratrol-induced apoptosis, whereas Smac N7 peptide induced apoptosis and enhanced the effectiveness of resveratrol.ConclusionResveratrol either alone or in combination with TRAIL or Smac can be used for the prevention and/or treatment of human prostate cancer.

Bax and Bak genes are essential for maximum apoptotic response by curcumin, a polyphenolic compound and cancer chemopreventive agent derived from turmeric, Curcuma longa

Curcumin, an active ingredient of turmeric (Curcuma longa), inhibits proliferation and induces apoptosis in cancer cells, but the sequence of events leading to cell death is poorly defined. The objective of this study was to examine the molecular mechanisms by which multidomain pro-apoptotic Bcl-2 family members Bax and Bak regulate curcumin-induced apoptosis using mouse embryonic fibroblasts (MEFs) deficient in Bax, Bak or both genes. Curcumin treatment resulted an increase in the protein levels of both Bax and Bak, and mitochondrial translocation and activation of Bax in MEFs to trigger drop in mitochondrial membrane potential, cytosolic release of apoptogenic molecules [cytochrome c and second mitochondria-derived activator of caspases (Smac)/direct inhibitor of apoptosis protein-binding protein with low isoelectric point], activation of caspase-9 and caspase-3 and ultimately apoptosis. Furthermore, MEFs derived from Bax and Bak double-knockout (DKO) mice exhibited even greater protection against curcumin-induced release of cytochrome c and Smac, activation of caspase-3 and caspase-9 and induction of apoptosis compared with wild-type MEFs or single-knockout Bax(-/-) or Bak(-/-) MEFs. Interestingly, curcumin treatment also caused an increase in the protein level of apoptosis protease-activating factor-1 in wild-type MEFs. Smac N7 peptide enhanced curcumin-induced apoptosis, whereas Smac siRNA inhibited the effects of curcumin on apoptosis. Mature form of Smac sensitized Bax and Bak DKO MEFs to undergo apoptosis by acting downstream of mitochondria. The present study demonstrates the role of Bax and Bak as a critical regulator of curcumin-induced apoptosis and over-expression of Smac as interventional approaches to deal with Bax- and/or Bak-deficient chemoresistant cancers for curcumin-based therapy.