Overexpression Of Smac Research Articles

Interaction of SMAC with a survivin-derived peptide alters essential cancer hallmarks: Tumor growth, inflammation, and immunosuppression

SMAC/Diablo is known as a pro-apoptotic mitochondrial protein released into the cytosol in response to apoptotic signals. We recently reported SMAC overexpression in cancers as essential for cell proliferation and tumor growth due to non-apoptotic functions including phospholipid synthesis regulation. These functions may be associated with its interactions with partner proteins. Using a peptide array with 768 peptides derived from 11 selected SMAC-interacting proteins, we identified SMAC-interacting sequences. These SMAC-binding sequences were produced as cell penetrating peptides targeted to the cytosol, mitochondria, or nucleus, inhibiting cell proliferation and inducing apoptosis in several cell lines. For in-vivo study, a survivin/BIRC5-derived peptide was selected due to its overexpression in many cancers, and involvement in mitosis, apoptosis, autophagy, cell proliferation, inflammation, and immune responses, as a target for cancer therapy. Specifically, a SMAC-targeting survivin/BIRC5-derived peptide, given intratumorally or intravenously, strongly inhibited lung tumor growth, cell proliferation, angiogenesis, and inflammation, induced apoptosis, and remodeled the tumor microenvironment. The peptide promoted tumor infiltration of CD8+ cells and increased cell-intrinsic PD-1/PD-L1 expression, resulting in cancer cell self-destruction and increased tumor cell death, preserving immune cells. Thus, targeting the interaction between the multifunctional proteins SMAC and survivin represents a innovative therapeutic cancer paradigm.

Co-overexpression of TRAIL and Smac sensitizes MDA-MB-231 cells to radiation through apoptosis depending on mitochondrial pathway

Pro-apoptosis in cancer cells has been proposed as a beneficial therapeutic strategy for potentiating the anticancer effects of radiotherapy. TNF-related apoptosis inducing ligand (TRAIL) and Second mitochondria derived activator of caspase (Smac) can induce cell apoptosis. Herein, we designed a conditionally replicating adenoviral co-overexpression vector of TRAIL and Smac regulated by the Egr1 promoter, in which hTERT, E1A-E1B and E1B55K genes were inserted to achieve enhanced tumor targeting characteristics. After breast cancer MDA-MB-231 cells were infected and irradiated, cellular proliferation and colony formation were measured, apoptotic rate was detected by FCM after AnnexinV-FITC/PI staining. To explore the molecular mechanisms of apoptosis, mRNA and protein levels of TRAIL, Smac, Cytochrome c (Cyt c), death receptor 5 (DR5), caspase-8, -9 and -3 were measured by quantitative real-time PCR, ELISA and Western blot, and caspase-3 activity was detected using caspase-3 activity kits. The results showed that TRAIL and/or Smac overexpression enhanced proliferation inhibition and radio-sensitivity through apoptosis. In addition, the combination of IR and overexpression of TRAIL and/or Smac can activate more apoptosis in tumor cells, and the transcriptional levels and protein expressions of Cyt c, DR5, caspase-8, -9 and -3 had similar regularity with apoptotic changes, indicating the molecular mechanisms of TRAIL and Smac involves the mitochondrial pathway. Our findings may have implications for novel radiotherapy plans for breast tumor treatment.

Overexpression of Smac is associated with the development of primary Sjogren's syndrome.

Primary Sjögren's syndrome (pSS) is a systemic autoimmune disease characterized by keratoconjunctivitis sicca, xerostomia, and extraglandular abnormalities. The precise etiology remains unclear. Previous studies have revealed that the apoptosis played an important role in the pSS. Herein, we investigated the expression and effect of second mitochondria-derived activator of caspase (Smac) in patients with pSS and associated the expression with clinicopathological parameters. Smac expression was checked in labial salivary glands of surgical specimens from cases of pSS using immunohistochemistry, reverse transcription-quantitative PCR, and Western blot. The results of immunohistochemistry were analyzed for clinicopathological parameters. In addition, the content of Smac in cytoplasm and mitochondria were examined. The mRNA of Smac, the content of Smac in cytoplasm, the Smac protein in the pSS patients increased significantly compared with the healthy controls (p < 0.05). The content of Smac in mitochondria decreased significantly compared with the healthy controls (p < 0.05). The integral optical density of Smac protein levels were positively correlated with IgG (r = 0.7435, p < 0.05) and erythrocyte sedimentation rate (ESR) (r = 0.7925, p < 0.05). Smac plays an important role in the development of pSS.

Overexpression of Smac by an Armed Vesicular Stomatitis Virus Overcomes Tumor Resistance

Despite reports of successful clinical cases, many tumors appear to resist infection by oncolytic viruses (OVs). To circumvent this problem, an armed vesicular stomatitis virus was constructed by inserting a transgene to express Smac/DIABLO during virus infection (VSV-S). Endogenous Smac in HeLa cells was diminished during wtVSV infection, whereas the Smac level was enhanced during VSV-S infection. Apoptosis was readily induced by VSV-S, but not wtVSV, infection. More importantly, the tumor volume was reduced to a larger extent when xenografts of 4T1 cells in BALB/c mice and OV-resistant T-47D cells in nude mice were intratumorally injected with VSV-S. VSV-S represents a novel mechanism to overcome tumor resistance, resulting in more significant tumor regression due to enhanced apoptosis.

Indomethacin induces apoptosis in the EC109 esophageal cancer cell line by releasing second mitochondria-derived activator of caspase and activating caspase-3.

The use of non‑steroidal anti‑inflammatory drugs (NSAIDs) has been associated with a reduced risk of various types of cancer, including esophageal cancer. However, the mechanisms underlying the antineoplastic effects of NSAIDs in esophageal cancer remain to be elucidated. In the present study, a significant inhibition in cell viability was observed in the EC109 cells following treatment with different concentrations of indomethacin, and these effects occurred in a dose‑ and time‑dependent manner. This inhibition was due to the release of second mitochondria‑derived activator of caspase (Smac) into the cytosol and the activation of caspase‑3. Subsequently, flow cytometry was performed to investigate indomethacin‑induced apoptosis following the overexpression or knockdown of Smac, and western blot analysis was performed to determine the expression of Smac and the activation of caspase‑3. Overexpression of Smac was promoted apoptosis, while downregulation of Smac significantly inhibited apoptosis. Western blot analysis demonstrated that indomethacin induced apoptosis through releasing Smac into the cytosol and activating caspase‑3. These results indicated that Smac is essential for the apoptosis induced by indomethacin in esophageal cancer cells.

Dual‑sensitive HRE/Egr1 promoter regulates Smac overexpression and enhances radiation‑induced A549 human lung adenocarcinoma cell death under hypoxia.

The aim of this study was to construct an expression vector carrying the hypoxia/radiation dual‑sensitive chimeric hypoxia response element (HRE)/early growth response1 (Egr‑1) promoter in order to overexpress the therapeutic second mitochondria‑derived activator of caspases (Smac). Using this expression vector, the present study aimed to explore the molecular mechanism underlying radiotherapy‑induced A549 human lung adenocarcinoma cell death and apoptosis under hypoxia. The plasmids, pcDNA3.1‑Egr1‑Smac (pE‑Smac) and pcDNA3.1‑HRE/Egr-1‑Smac (pH/E‑Smac), were constructed and transfected into A549 human lung adenocarcinoma cells using the liposome method. CoCl2 was used to chemically simulate hypoxia, followed by the administration of 2Gy X‑ray irradiation. An MTT assay was performed to detect cell proliferation and an Annexin V‑fluorescein isothiocyanate apoptosis detection kit was used to detect apoptosis. Quantitative polymerase chain reaction and western blot analyses were used for the detection of mRNA and protein expression, respectively. Infection with the pE‑Smac and pH/E‑Smac plasmids in combination with radiation and/or hypoxia was observed to enhance the expression of Smac. Furthermore, Smac overexpression was found to enhance the radiation‑induced inhibition of cell proliferation and promotion of cycle arrest and apoptosis. The cytochromec/caspase‑9/caspase‑3 pathway was identified to be involved in this regulation of apoptosis. Plasmid infection in combination with X‑ray irradiation was found to markedly induce cell death under hypoxia. In conclusion, the hypoxia/radiation dual‑sensitive chimeric HRE/Egr‑1 promoter was observed to enhance the expression of the therapeutic Smac, as well as enhance the radiation‑induced inhibition of cell proliferation and promotion of cycle arrest and apoptosis under hypoxia. This apoptosis was found to involve the mitochondrial pathway.

Overexpression of Smac Promotes Cisplatin-Induced Apoptosis by Activating Caspase-3 and Caspase-9 in Lung Cancer A549 Cells

Second mitochondrial-derived activator of caspase (Smac) plays crucial roles in mitochondrial apoptosis pathways and promotes chemotherapy-induced apoptosis. In this study, Smac levels were examined in various lung cancer cell lines, and the effects of overexpressed Smac in the nonsmall-cell lung cancer cell line A549 were assayed by stable transfection of Smac. Subsequently, MTT assays, cell counting, and flow cytometry were applied to show that overexpression of Smac inhibits cell viability and cell growth and enhances apoptosis after cisplatin treatment. Western blotting was performed before and after cisplatin treatment to demonstrate that drug treatment could release Smac from mitochondria into the cytosol and promote apoptosis by activating caspase-3 and caspase-9. Promotion of apoptosis by cytosolic Smac could be blocked by pretreating cells with the caspase-9 inhibitor z-LEHD-FMK. Our findings indicate that overexpressed Smac significantly inhibited A549 cell growth and promoted apoptosis following cisplatin treatment due to the release of Smac from mitochondria into the cytosol, which increased the activities of caspase-3 and caspase-9.

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.

Smac Modulates Chemosensitivity in Head and Neck Cancer Cells through the Mitochondrial Apoptotic Pathway

Overexpression of inhibitors of apoptosis proteins (IAP) contributes to therapeutic resistance. Second mitochondria-derived activator of caspase (Smac) promotes caspase activation by binding to IAPs upon release from the mitochondria. IAP antagonists, also called SMAC mimetics, are promising anticancer agents modeled after this mechanism. We investigated the role and mechanisms of Smac- and Smac mimetic-mediated chemosensitization in head and neck squamous cell carcinoma (HNSCC) cells. The effects of SMAC knockdown, SMAC overexpression, and a small molecule Smac mimetic on the chemosensitivities of HNSCC cells were determined. The mechanisms of Smac- and Smac mimetic-mediated chemosensitization were investigated by analyzing growth suppression, the mitochondrial apoptotic pathway, caspase activation, and IAP proteins. The therapeutic responses of HNSCC cells with different levels of Smac were compared in xenograft models. We found that Smac mediates apoptosis induced by several classes of therapeutic agents through the mitochondrial pathway. SMAC knockdown led to impaired caspase activation, mitochondrial membrane depolarization, and release of cytochrome c. A small molecule Smac mimetic, at nanomolar concentrations, significantly sensitized HNSCC cells to gemcitabine-induced apoptosis and restored gemcitabine sensitivity in SMAC knockdown cells, through caspase activation, X-linked IAP dissociation, and mitochondria-associated events, but not the TNF-α pathway. Furthermore, Smac levels modulated the therapeutic response of HNSCC cells to gemcitabine in xenograft models. Our results establish a critical role of Smac in mediating therapeutic responses of HNSCC cells and provide a strong rationale for combining Smac mimetics with other anticancer agents to treat HNSCC.

Knockdown of Second Mitochondria-Derived Activator of Caspase Expression by RNAi Enhances Growth and Cisplatin Resistance of Human Lung Cancer Cells

Second mitochondria-derived activator of caspase (Smac) is a mitochondrial protein that promotes apoptosis in many kinds of cancers. Here, for the first time, the effects of Smac RNAi on growth and drug resistance to cisplatin [cis-diamminedichloroplatinum(II)] of lung cancer cells are investigated. Knockdown of Smac expression in A549 and 95D cells was mediated by transfection with pGC-FU vector containing siRNA sequences targeting human Smac with the lentivirus vector system. Smac was also overexpressed by transfection with pOE vector containing full-length coding region of Smac. Cell growth, cell cycle, and apoptosis were measured by methyl-thiazol tetrazolium assay, colony-formation assay, and flow cytometry. Drug resistance was performed by treatment with 10 μg/mL cisplatin. Downregulation of Smac enhanced cell growth and drug resistance to cisplatin of A549 and 95D cells, whereas overexpression of Smac did reversely. Smac helps inhibit cell growth and potentiate drug sensitivity to cisplatin of lung cancer cells.

Abstract 1218: SMAC modulates gemcitabine-induced apoptosis in head and neck cancer cells

Abstract Apoptosis induction is an important anti-tumor mechanism of many conventional chemotherapeutic agents. Biochemical data indicate that SMAC promotes caspase activation by antagonizing IAPs. However, SMAC-knockout mice do not exhibit obvious apoptotic deficiencies in response to a wide range of stimuli. Work for us and others demonstrate that SMAC plays an essential role in apoptosis induced by Nonsteroidal Anti-inflammatory Drug (NSAID), TRAIL or BH3 only proteins in colon cancer cells. In this study we documented a critical role of SMAC in mediating apoptosis induced by chemotherapeutic agent gemcitabine in head and neck squamous cell carcinoma (HNSCC) cells. Using RNA interference, we found that that SMAC mediates gemcitabine-induced apoptosis through the intrinsic/mitochondrial pathway. SMAC Knockdown led to impairment in caspase-3 activation, mitochondrial membrane depolarization and release of cytochrome c. Small-molecule SMAC mimetics at nanomolar concentrations significantly sensitized HNSCC cells to gemcitabine-induced apoptosis, and overcame gemcitabine resistance in SMAC-knockdown cells by restoring caspase-3 activation and cytochrome c release. Furthermore, in xenograft models, SMAC knockdown significantly blocked the therapeutic responses of HNSCC tumors to gemcitabine by inhibiting apoptosis, while SMAC overexpression dramatically enhanced that. Together, these results suggest that SMAC mediates apoptosis in cancer cells, and provide a strong rationale for combining SMAC mimetics with other anticancer agents to treat apoptosis-resistant tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1218.

Apoptosis Induced by Extracts of Helleborus Niger in Different Lymphoma and Leukemia Cell Lines and Primary Lymphoblasts of Children with ALL Is Independent of Smac-Overexpression and Executed Via the Mitochondrial Pathway.

Helleborus niger, also known as Christmas Rose, belongs to the family of Ranunculaceae, a family of flowering plants with about 2500 different species. In complementary medicine Helleborus niger is used as adjuvant drug in the treatment of non-metastasised and metastasised forms of bronchial cancer, abdominal tumours and prostate cancer. It is also applied in myeloproliferative diseases like Hodgkin and Non-Hodgkin lymphoma, leukaemic disorders and AIDS- related diseases like the Kaposi sarcoma. Until now, there is no clinical or preclinical data regarding the effects of Helleborus niger in vivo, ex vivo or in vitro. For this purpose, we investigated the cytotoxic effects of four different standardized aqueous Helleborus niger extracts from the companies Hiscia and Helixor on various cancer cell lines. We used one whole plant extract, one root extract, one leave extract and one containing only the blossom of Helleborus niger. After 4h of treatment with the extracts no significant LDH release was measured, thus excluding an unspecific, necrotic damage of the cell membrane. After 24h a dose dependent inhibition of proliferation up to 69% could be found and after 48h a distinction into early (45,2%) and late apoptotic (45,5%) cells was detected via Annexin/PI staining. The cell cycle analysis revealed characteristic hypodiploid DNA fragments after 72h, once more identifying apoptosis as cause of the cell death. In the Western Blot analysis a processing of Caspase-3 could be found after 36 h incubation with the extract. Apoptotic cell death was detected in the Burkitt-like lymphoma cell line BJAB, the three human acute lymphoblastic leukemia cell lines NALM-6, Sup-B-15 and REH and the melanoma cell line MEL-HO. The apoptosis induction caused by the root extract was higher than the apoptotic cell death in the other extracts. There are two major pathways of apoptosis, the extrinsic pathway via death receptors like FADD and the intrinsic pathway via the mitochondria. In BJAB cells a breakdown of the mitochondrial membrane potential and dose-dependent mitochondrial permeability transition was detected after 48h, revealing that apoptosis is executed via the mitochondrial pathway. Furthermore, we found a decreased apoptosis induction in BCL-2 overexpressing melanoma cells. The dependency of Bcl-2 expression is another sign of apoptosis via the mitochondrial pathway. In contrast, apoptosis induction by Helleborus niger seems to be independent of Smac overexpression, which could be shown in Jurkat cells. In combination with the vinca alkaloid vincristine, which is used in the treatment of ALL, a synergistic effect could be detected. The apoptosis induction was up to 16% higher in combination than in the single treatment. Finally, we evaluated the effect on primary leukemia cells ex vivo. Interestingly, we could show a significant apoptosis induction in primary leukemia cells from 2 patients with ALL or AML in childhood, which were resistant to the treatment with the anthracycline doxorubicin. For the first time, we were able to show that extracts of Helleborus niger induce apoptosis in different cancer cell lines and primary leukemia cells. Apoptosis is executed via the intrinsic pathway and is independent of Smac overexpression. Thus, we present an interesting baseline for the design of upcoming in vivo experiments or clinical trials.

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.

Altered Expression of c-IAP1, Survivin, and Smac Contributes to Chemotherapy Resistance in Thyroid Cancer Cells

Resistance to chemotherapy predicts an unfavorable outcome for patients with radioiodine-insensitive thyroid cancer. To investigate the mechanisms underlying this resistance, we evaluated the expression of four different inhibitor of apoptosis proteins, and their antagonist, Smac, in thyroid cancer cells that survived 48 hours of exposure to cisplatin, doxorubicin, or taxol. We found high levels of c-IAP1 after cisplatin treatment and increased expression of survivin following exposure to doxorubicin. Cells that endured treatment with taxol showed reduced expression of Smac and released minimal amounts of this protein from the mitochondria. Down-regulation of c-IAP1 and survivin increased the cytotoxicity of cisplatin and doxorubicin, whereas overexpression of Smac improved the efficacy of taxol. Finally, thyroid cancer cells permanently resistant to doxorubicin or cisplatin showed increased expression of c-IAP1 and survivin, respectively. However, silencing of these proteins by RNA interference restored sensitivity to doxorubicin and cisplatin. Thus, in thyroid cancer cells, early resistance to chemotherapeutic agents requires high levels of c-IAP1 and survivin and low levels of Smac. Furthermore, increased expression of c-IAP1 and survivin contributes to the acquisition of permanent resistance to cytotoxic compounds.

Regulatory Effects of X-linked Inhibitor of Apoptosis Protein and Pro-apoptotic Protein Smac on Apoptosis Resistance to Chemotherapy in Pancreatic Cancer Cells

Objective: To investigate the relation of X-linked inhibitor of apoptosis (XIAP) and second mitochondria-derived activator of caspase (Smac) signaling pathway to chemoresistance in human pancreatic cancer Panc-1 and BXPC-3 cells. Methods: Apoptosis and the changes of XIAP expression in permeabilized cells induced by cisplatin and 5-fluorouracil (FU) were measured by flow cytometry. The cytosolic expression of XIAP, Smac and caspase-3 was detected by Western blot. A recombinant plasmid vector pEGFP-N1/Smac was constructed and transfected into of Panc-1 cells. The effect of cytosolic overexpression of Smac on apoptosis of Panc-1 cells was evaluated by flow cytometry. Results: Panc-1 was more resistant to cisplatin or 5-FU induced apoptosis than BXPC-3. Western blot revealed that chemoresistant Panc-1 highly expressed XIAP, and increased cytosolic expression of Smac might be responsible for the marked down-regulation of XIAP in chemo-sensitive BXPC-3 cells after exposure to cisplatin or 5-FU. Furthermore, cytosolic overexpression of Smac could significantly down-regulate the levels of XIAP and promote the activity of caspase-3, as well as sensitize Panc-1 cells to anticancer drug-induced apoptosis. Conclusion: Anticancer drug-induced apoptosis requires mitochondrial release of Smac and downregulation of XIAP, which may be an important determinant of chemo-sensitivity in pancreatic cancer cells. Up-regulation of cytosolic expression of Smac may act as an effective modifying signal to overcome apoptosis resistance to chemotherapy in pancreatic cancer cells.

Transfection of Smac sensitizes tumor cells to etoposide-induced apoptosis and eradicates established human hepatoma in vivo

A major concern in clinical treatment of cancers is resistance of tumors such as hepatocellular carcinoma (HCC) and osteosarcoma to current chemotherapy protocols. Here, we reported that overexpression of second mitochondria-derived activator of caspase (Smac) sensitized osteosarcoma cells and HCC cells in vitro to chemotherapeutic drugs-induced apoptosis. Constitutive expression of Smac resulted in enhanced Bax accumulation on mitochondria upon etoposide stimulation and inhibited Bcl-2-induced antiapoptosis activity. Thus, Smac would sensitize tumor cells to chemotherapeutic drugs in part through promoting Bax translocation to mitochondria and bypassing Bcl-2 block. Moreover, we demonstrated that blockade of Smac expression by antisense smac did not impair etoposide-induced apoptosis; however, p53-induced apoptosis was impaired in smac deficient Saos-2 cell. This suggested Smac might be required in p53-induced apoptosis. Most importantly, complete eradication of HepG2 xenografts in vivo was achieved upon combined therapy with Ad-Smac and 5-Fu. Thus, overexpression of Smac in tumor cells might be a potent strategy for cancer treatment by sensitization of tumor cells to chemotherapeutic drugs.

Regulatory effects on apoptotic activities of gastric cancer cell line by over-expression of Smac gene

To investigate the effects of overexpression of second mitochondria-derived activator of caspases (Smac) gene on apoptosis of gastric cancer cells. Under the induction of liposome, MKN-45 cells were transfected by Smac gene and incubated with G418 for subclone selection. Reverse transcriptase-polymerase chain reaction and Western blot were used to determine cellular Smac gene expression. After induction of apoptosis by mitomycin (MMC), cell viabilities were analyzed using trypan blue stain. Apoptosis was measured by electronic microscopy, acridine orange-ethidium bromide fluorescent staining and in situ terminally labelled transferase technique (TUNEL). Western blot and colorimetry were used to assess cellular caspase-3 expression and its activity. The Smac mRNA and protein levels in MKN-45/Smac subclone cells (subclone consistently expressing Smac gene) were significantly higher than those in MKN-45 (P < 0.01). When compared with those in MKN-45, cell viabilities of MKN-45/Smac were reduced by 10.0% to 30.8% (P < 0.01), after treatment with 10 microg/ml MMC for 6 to 24 hours. Some of the MKN-45/Smac cells showed characteristic morphologic changes of apoptosis, their apoptotic rate being increased by 21.2% (P < 0.01). After treatment with MMC, caspase-3 expression and its activity in MKN-45/Smac cells were significantly higher than those in MKN-45 (P < 0.01). Overexpression of Smac in gastric cancer cell line significantly improves expression and activity levels of caspase-3 after induction by MMC. Such apoptosis-inducing effect establishes a novel strategy for regulating the apoptosis activity of gastric cancer.

Human colon cancer cells lacking Bax resist curcumin-induced apoptosis and Bax requirement is dispensable with ectopic expression of Smac or downregulation of Bcl-XL

Multiple apoptotic stimuli induce conformational changes in Bax, a proapoptotic protein from the Bcl-2 family and its deficiency is a frequent cause of chemoresistance in colon adenocarcinomas. Curcumin, a dietary compound from turmeric, is known to induce apoptosis in a variety of cancer cells. To understand the role of Bax in curcumin-induced apoptosis we used HCT116 human colon cancer cells with one allele of Bax gene (Bax+/-) and Bax knockout HCT116 (Bax-/-) cells in which Bax gene is inactivated by homologous recombination. Cell viability decreased in a concentration-dependent manner in Bax+/- cells treated with curcumin (0-50 microM) whereas only minimal changes in viability were observed in Bax-/- cells upon curcumin treatment. In Bax-/- cells curcumin-induced activation of caspases 9 and 3 was blocked and that of caspase 8 remained unaltered. Curcumin-induced release of cytochrome c, Second mitochondria derived activator of caspase (Smac) and apoptosis inducing factor (AIF) was also blocked in Bax-/- cells and reintroduction of Bax, downregulation of the antiapoptotic protein Bcl-XL by antisense DNA as well as the overexpression of Smac, highly sensitized the Bax-/- cells toward curcumin-induced apoptosis. There was no considerable difference in the percentage of apoptotic cells in Bak RNAi transfected Bax+/- or Bax-/- cells treated with curcumin when compared with their corresponding vector transfected cells treated with curcumin. The present study demonstrates the role of Bax but not Bak as a critical regulator of curcumin-induced apoptosis and implies the potential of targeting antiapoptotic proteins like Bcl-XL or overexpression of proapoptotic proteins like Smac as interventional approaches to deal with Bax-deficient chemo-resistant cancers for curcumin-based therapy.

The Membrane-associated Inhibitor of Apoptosis Protein, BRUCE/Apollon, Antagonizes Both the Precursor and Mature Forms of Smac and Caspase-9

Smac/DIABLO, HtrA2/Omi, and caspase-9 play key roles in the initiation of apoptosis. The inhibitor of apoptosis proteins (IAPs) are believed to bind to the N-terminal IAP binding motifs of the mature (proteolytically processed) forms of Smac, HtrA2, and caspase-9. However, we show here that BRUCE/Apollon, a 528-kDa IAP whose degradation promotes apoptosis, associates with their precursors as well as the mature forms by binding to regions in addition to the IAP binding motif. Through these associations, BRUCE promotes the degradation of Smac and inhibits the activity of caspase-9 but not the effector caspase, caspase-3. In response to apoptotic stimuli, BRUCE is degraded by proteasomes and/or cleaved by caspases and HtrA2 depending on the specific stimulus and the cell type. These results suggest that the ability of BRUCE to antagonize both the precursor and mature forms of Smac and caspase-9 is an important mechanism for the prevention of apoptosis under normal conditions.

Sensitization for γ-Irradiation-Induced Apoptosis by Smac.

Smac is released from mitochondria during the onset of apoptosis and promotes apoptosis via abrogating the binding of Inhibitor of Apoptosis Proteins (IAPs) to caspases. γ-irradiation is one of the most commonly used therapeutic approaches in clinical oncology, which triggers cell death in tumors via DNA and/or membrane damage. Since we recently found that Smac agonists sensitized even resistant tumors for apoptosis induced by death receptor ligation or anticancer drugs, we investigated the effect of Smac agonists on apoptosis following γ-irradiation in the present study. Here, we report for the first time that overexpression of mitochondrial or cytosolic Smac significantly increased radiosensitivity of various cancers.Transfection-enforced expression of Smac strongly enhanced apoptosis upon γ-irradiation in SH-EP neuroblastoma cells, which were resistant to g-irradiation in the absence of Smac. Importantly, Smac overexpression also reduced clonogenic tumor cell survival following γ-irradiation. Analysis of signaling pathways revealed that overexpression of Smac resulted in more rapid and more potent activation of caspase pathways, e.g caspase-2, -3,- 8, -9. The broad range caspase inhibitor zVAD.fmk abrogated apoptosis upon γ-irradiation indicating that apoptosis was mediated by caspases. In addition, overexpression of Smac promoted loss of mitochondrial membrane potential and cytochrome c release upon γ-irradiation. Interestingly, γ-irradiation-induced mitochondrial perturbations were blocked in the presence of the caspase inhibitor zVAD.fmk suggesting that caspase activity was required for mitochondrial alterations in response to γ-irradiation. Notably, cell cycle alterations and activation of NF-κB occured in a similar manner in vector control and Smac-transfected cells suggesting that Smac did not significantly alter the initial cellular stress response upon γ-irradiation. Importantly, Smac overexpression sensitized various tumor cell lines for γ-irradiation-induced apoptosis, indicating that the sensitization effect of Smac for γ-irradiation was not restricted to a particular cell type. By demonstrating that Smac can sensitize various tumor cells towards γ-irradiation-induced cell death, our findings provide for the first time evidence that Smac agonists may be a useful tool to enhance radiosensitivity in a variety of human cancers.