BACKGROUND: Recent discoveries in cancer therapeutics have proven combination therapies more effective than individual drugs. This study describes the efficacy of the combination of Cinnamomum zeylanicum and doxorubicin against benzene-induced leukemia. METHODS AND RESULTS: Brine shrimp assay was used to assess the cytotoxicity of C. zeylanicum, doxorubicin and their combination. After AML induction in Sprague Dawley rats, the same drugs were given to rat groups. Changes in organ weight, haematological profile, and hepatic enzymes were determined. Real-time PCR was used to elucidate the effect on the expression of STMN1, GAPDH, P53 and various TRAIL and NF-kappaB components. C. zeylanicum reduced the cytotoxicity of doxorubicin. The combination treatment showed better anti-leukemic results than any of the individual drugs as evident from STMN1 expression (p < 0.001). It was particularly effective in reducing total white blood cell counts and recovering lymphocytes, monocytes and eosinophils along with hepatic enzymes ALT and AST (p < 0.001). All doses recovered relative organ weights and improved blood parameters. The combination therapy was particularly effective in inducing apoptosis, inhibition of proliferation marker GAPDH (p < 0.001) and NF-kappaB pathway components Rel-A (p < 0.001) and Rel-B (p < 0.01). Expressions of TRAIL components c-FLIP (p < 0.001), TRAIL ligand (p < 0.001) and caspase 8 (p < 0.01) were also altered. CONCLUSION: Cinnamomum zeylanicum in combination with doxorubicin helps to counter benzene-induced cellular and hepatic toxicity and improves haematological profile. The anti-leukemic effects are potentially due to inhibition of GAPDH and NF-kappa B pathway, and through regulation of TRAIL pathway. Our data suggests the use of C. zeylanicum with doxorubicin to improve anti-leukemic therapeutic regimes.
- MeSH
- apoptóza MeSH
- benzen farmakologie MeSH
- doxorubicin farmakologie MeSH
- krysa rodu rattus MeSH
- leukemie * farmakoterapie MeSH
- NF-kappa B metabolismus MeSH
- oleje prchavé * farmakologie MeSH
- potkani Sprague-Dawley MeSH
- protein TRAIL metabolismus farmakologie MeSH
- skořicovník ceylonský metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
OBJECTIVES: Renal cell carcinoma (RCC) is the most lethal urologic malignancy with increasing incidence worldwide. The conventional treatment strategies for advanced or recurrent RCC are not efficient and show considerable toxicities. Adoptive cell transfer (ACT) has become a promising treatment option for multiple cancers, particularly in combination with other therapeutic approaches. ACT often utilizes extensively in vitro expanded tumor-infiltrating lymphocytes (TILs). However, TILs are a very heterogeneous mix of cell populations and only those populations that have a cytotoxic and migratory potential are thought to deliver a therapeutic impact in ACT. The identification and localization of these therapeutically potent populations are therefore needed. METHODS AND MATERIALS: A total number of 57 tissue samples from 19 RCC patients who underwent radical nephrectomy was analyzed. The tissue samples were obtained from the tumor, peritumoral tissue, and the adjacent healthy renal tissue. The tissues were sliced, enzymatically dissociated into single cell suspensions and the obtained cells further analyzed by flow cytometry for the expression of markers of lymphocyte cytotoxicity - TRAIL and FasL, and a surrogate marker of lymphocyte migratory activity - PECAM-1. The analyzed data were next correlated with the clinical and histopathological data. RESULTS: Non-clear cell RCC (non-ccRCC) tumors showed a significantly decreased tumor infiltration with TRAIL+FasL+ NK cells but elevated infiltration with FasL+PECAM-1+ T cells as compared with clear cell RCC (ccRCC) tumors. Further analyses revealed that the peritumoral tissue of ccRCC patients is a reservoir of TRAIL+FasL+, TRAIL+PECAM-1+, or FasL+PECAM-1+ NK and T cells. CONCLUSIONS: The cytotoxic/migratory lymphocytes were identified in tumors of ccRCC patients. These lymphocytes became excluded from the tumor and accumulated in the patient's peritumoral tissue.
- MeSH
- antigeny CD31 metabolismus MeSH
- buňky NK imunologie metabolismus MeSH
- cytotoxicita imunologická MeSH
- karcinom z renálních buněk imunologie patologie chirurgie MeSH
- ledviny cytologie imunologie patologie chirurgie MeSH
- lidé středního věku MeSH
- lidé MeSH
- ligand Fas metabolismus MeSH
- nádory ledvin imunologie patologie chirurgie MeSH
- nefrektomie MeSH
- protein TRAIL metabolismus MeSH
- průtoková cytometrie MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- separace buněk MeSH
- T-lymfocyty imunologie metabolismus MeSH
- tumor infiltrující lymfocyty imunologie metabolismus MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- pozorovací studie MeSH
- práce podpořená grantem MeSH
Background: Apoptosis plays an important role in the myocardial injury after acute myocardial infarction and in the subsequent development of heart failure. Aim: To clarify serum kinetics of apoptotic markers TRAIL and sFas and their relation to left ventricular ejection fraction (LVEF) in patients with ST-elevation myocardial infarction (STEMI) treated with primary percutaneous coronary intervention (pPCI). Methods: In 101 patients with STEMI treated with pPCI, levels of TRAIL and sFas were measured in series of serum samples obtained during hospitalization and one month after STEMI. LVEF was assessed at admission and at one month. Major adverse cardiovascular events (MACE, i.e., death, re-MI, and hospitalization for heart failure and stroke) were analysed during a two-year followup. Results: Serum level of TRAIL significantly decreased one day after pPCI (50.5pg/mL) compared to admission (56.7pg/mL), subsequently increased on day 2 after pPCI (58.8pg/mL), and reached its highest level at one month (70.3pg/mL). TRAIL levels on days 1 and 2 showed a significant inverse correlation with troponin and a significant positive correlation with LVEF at baseline. Moreover, TRAIL correlated significantly with LVEF one month after STEMI (day 1: r=0.402, p<0.001; day 2: r=0.542, p<0.001). On the contrary, sFas level was significantly lowest at admission (5073pg/mL), increased one day after pPCI (6370pg/mL), and decreased on day 2 (5548pg/mL). Significantly highest sFas level was marked at one month (7024pg/mL). sFas failed to correlate with LVEF at baseline or at one month. Both TRAIL and sFas showed no ability to predict improvement of LVEF one month after STEMI or a 2-year MACE (represented by 3.29%). Conclusion: In STEMI treated with pPCI, TRAIL reaches its lowest serum concentration after reperfusion. Low TRAIL level is associated with worse LVEF in the acute phase of STEMI as well as one month after STEMI. Higher TRAIL level appears to be beneficial and thus TRAIL seems to represent a protective mediator of post-AMI injury.
- MeSH
- biologické markery metabolismus MeSH
- infarkt myokardu s elevacemi ST úseků patofyziologie terapie MeSH
- infarkt myokardu MeSH
- koronární angioplastika * MeSH
- lidé středního věku MeSH
- lidé MeSH
- protein TRAIL metabolismus MeSH
- senioři MeSH
- stenty uvolňující léky MeSH
- tepový objem * MeSH
- výsledek terapie MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Purpose: One third of ER-positive breast cancer patients who initially respond to endocrine therapy become resistant to treatment. Such treatment failure is associated with poor prognosis and remains an area of unmet clinical need. Here, we identify a specific posttranslational modification that occurs during endocrine resistance and which results in tumor susceptibility to the apoptosis-inducer TRAIL. This potentially offers a novel stratified approach to targeting endocrine-resistant breast cancer.Experimental Design: Cell line and primary-derived xenograft models of endocrine resistance were investigated for susceptibility to TRAIL. Tumor viability, cancer stem cell (CSC) viability (tumorspheres), tumor growth kinetics, and metastatic burden were assessed. Western blots for the TRAIL-pathway inhibitor, c-FLIP, and upstream regulators were performed. Results were confirmed in primary culture of 26 endocrine-resistant and endocrine-naïve breast tumors.Results: Breast cancer cell lines with acquired resistance to tamoxifen (TAMR) or faslodex were more sensitive to TRAIL than their endocrine-sensitive controls. Moreover, TRAIL eliminated CSC-like activity in TAMR cells, resulting in prolonged remission of xenografts in vivo In primary culture, TRAIL significantly depleted CSCs in 85% endocrine-resistant, compared with 8% endocrine-naïve, tumors, whereas systemic administration of TRAIL in endocrine-resistant patient-derived xenografts reduced tumor growth, CSC-like activity, and metastases. Acquired TRAIL sensitivity correlated with a reduction in intracellular levels of c-FLIP, and an increase in Jnk-mediated phosphorylation of E3-ligase, ITCH, which degrades c-FLIP.Conclusions: These results identify a novel mechanism of acquired vulnerability to an extrinsic cell death stimulus, in endocrine-resistant breast cancers, which has both therapeutic and prognostic potential. Clin Cancer Res; 24(10); 2452-63. ©2018 AACR.
- MeSH
- antitumorózní látky hormonální farmakologie MeSH
- apoptóza účinky léků MeSH
- chemorezistence * genetika MeSH
- FLIP (buněčný) genetika metabolismus MeSH
- lidé MeSH
- modely nemocí na zvířatech MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nádorové kmenové buňky účinky léků metabolismus MeSH
- nádory prsu farmakoterapie genetika metabolismus MeSH
- posttranslační úpravy proteinů * MeSH
- protein TRAIL metabolismus farmakologie MeSH
- receptory pro estrogeny metabolismus MeSH
- regulace genové exprese u nádorů účinky léků MeSH
- xenogenní modely - testy antitumorózní aktivity MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Searching for new strategies for effective elimination of human prostate cancer cells, we investigated the cooperative cytotoxic action of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and two platinum-based complexes, cisplatin or LA-12, and related molecular mechanisms. We demonstrated a notable ability of cisplatin or LA-12 to enhance the sensitivity of several human prostate cancer cell lines to TRAIL-induced cell death via an engagement of mitochondrial apoptotic pathway. This was accompanied by augmented Bid cleavage, Bak activation, loss of mitochondrial membrane potential, activation of caspase-8, -10, -9, and -3, and XIAP cleavage. RNAi-mediated silencing of Bid or Bak in Bax-deficient DU 145 cells suppressed the drug combination-induced cytotoxicity, further underscoring the involvement of mitochondrial signaling. The caspase-10 was dispensable for enhancement of cisplatin/LA-12 and TRAIL combination-induced cell death and stimulation of Bid cleavage. Importantly, we newly demonstrated LA-12-mediated enhancement of TRAIL-induced cell death in cancer cells derived from human patient prostate tumor specimens. Our results provide convincing evidence that employing TRAIL combined with cisplatin/LA-12 could contribute to more effective killing of prostate cancer cells compared to the individual action of the drugs, and offer new mechanistic insights into their cooperative anticancer action.
- MeSH
- amantadin analogy a deriváty farmakologie MeSH
- apoptóza účinky léků MeSH
- cisplatina farmakologie MeSH
- kaspasa 10 metabolismus MeSH
- lidé MeSH
- mitochondrie účinky léků metabolismus MeSH
- nádory prostaty metabolismus patologie MeSH
- organoplatinové sloučeniny farmakologie MeSH
- protein Bid metabolismus MeSH
- protein TRAIL metabolismus MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
Despite recent advances in targeted therapeutics, administration of 5-fluorouracil (5-FU) remains a common clinical strategy for post-surgical treatment of solid tumors. Although it has been proposed that RNA metabolism is disturbed by 5-FU treatment, the key cytotoxic response is believed to be enzymatic inhibition of thymidylate synthase resulting in nucleotide pool disproportions. An operating p53 tumor suppressor signaling network is in many cases essential for the efficiency of chemotherapy, and malfunctions within this system remain a clinical obstacle. Since the fate of chemotherapy-insensitive tumor cells is rarely described, we performed a comparative analysis of 5-FU toxicity in p53-deficient cells and conclude that p53 acts as a facilitator rather than a gatekeeper of cell death. Although p53 can act as a regulator of several cellular stress responses, no rerouting of cell death mode was observed in absence of the tumor suppressor. Thus, the final death outcome of 5-FU-treated p53-/- cells is demonstrated to be caspase-dependent, but due to a slow pace, accumulation of mitochondrial reactive oxygen species contributes to necrotic characteristics. The oligomerization status of the p53 target gene DR5 is determined as a significant limiting factor for the initiation of caspase activity in an intracellular TRAIL-dependent manner. Using several experimental approaches, we further conclude that RNA-rather than DNA-related stress follows by caspase activation irrespectively of p53 status. A distinct 5-FU-induced stress mechanism is thereby functionally connected to a successive and discrete cell death signaling pathway. Finally, we provide evidence that silencing of PARP-1 function may be an approach to specifically target p53-deficient cells in 5-FU combinatorial treatment strategies. Together, our results disclose details of impaired cell death signaling engaged as a consequence of 5-FU chemotherapy. Obtained data will contribute to the comprehension of factors restraining 5-FU efficiency, and by excluding DNA as the main stress target in some cell types they propose alternatives to currently used and suggested synergistic treatment regimens.
- MeSH
- antimetabolity antitumorózní farmakologie MeSH
- apoptóza účinky léků MeSH
- fluorescenční protilátková technika MeSH
- fluorouracil farmakologie MeSH
- imunoblotting MeSH
- imunoprecipitace MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádorový supresorový protein p53 metabolismus MeSH
- polymerázová řetězová reakce MeSH
- protein TRAIL metabolismus MeSH
- RNA účinky léků MeSH
- signální transdukce účinky léků fyziologie MeSH
- transdukce genetická MeSH
- transmisní elektronová mikroskopie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In search for novel strategies in colon cancer treatment, we investigated the unique ability of platinum(IV) complex LA-12 to efficiently enhance the killing effects of tumor necrosis factor-related apoptosis inducing ligand (TRAIL), and compared it with the sensitizing action of cisplatin. We provide the first evidence that LA-12 primes human colon cancer cells for TRAIL-induced cytotoxicity by p53-independent activation of the mitochondrial apoptotic pathway. The cooperative action of LA-12 and TRAIL was associated with stimulation of Bax/Bak activation, drop of mitochondrial membrane potential, caspase-9 activation, and a shift of the balance among Bcl-2 family proteins in favor of the pro-apoptotic members. In contrast to cisplatin, LA-12 was a potent inducer of ERK-mediated Noxa and BimL protein upregulation, and more effectively enhanced TRAIL-induced apoptosis in the absence of Bax. The cooperative action of LA-12 and TRAIL was augmented following the siRNA-mediated silencing of Mcl-1 in both Bax proficient/deficient cells. We newly demonstrated that LA-12 induced ERK-mediated c-Myc upregulation, and proved that c-Myc silencing inhibited the mitochondrial activation and apoptosis in colon cancer cells treated with LA-12 and TRAIL. The LA-12-mediated sensitization to TRAIL-induced apoptosis was demonstrated in several colon cancer cell lines, further underscoring the general relevance of our findings. The selective action of LA-12 was documented by preferential priming of cancer but not normal colon cancer cells to TRAIL killing effects. Our work highlights the promising potential of LA-12 over cisplatin to enhance the colon cancer cell sensitivity to TRAIL-induced apoptosis, and provides new mechanistic insights into their cooperative action.
- MeSH
- amantadin analogy a deriváty farmakologie MeSH
- antitumorózní látky farmakologie MeSH
- apoptóza účinky léků genetika MeSH
- cisplatina farmakologie MeSH
- geny p53 MeSH
- HCT116 buňky účinky léků MeSH
- lidé MeSH
- membránový potenciál mitochondrií účinky léků MeSH
- mitochondrie účinky léků metabolismus MeSH
- nádorové buňky kultivované MeSH
- nádory tračníku farmakoterapie metabolismus patologie MeSH
- organoplatinové sloučeniny farmakologie MeSH
- protein Bak metabolismus MeSH
- protein TRAIL metabolismus farmakologie MeSH
- protein X asociovaný s bcl-2 genetika metabolismus MeSH
- protoonkogenní proteiny c-bcl-2 metabolismus MeSH
- protoonkogenní proteiny c-myc genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Death ligands and their tumor necrosis factor receptor (TNFR) family receptors are the best-characterized and most efficient inducers of apoptotic signaling in somatic cells. In this study, we analyzed whether these prototypic activators of apoptosis are also expressed and able to be activated in human pluripotent stem cells. We examined human embryonic stem cells (hESC) and human-induced pluripotent stem cells (hiPSC) and found that both cell types express primarily TNF-related apoptosis-inducing ligand (TRAIL) receptors and TNFR1, but very low levels of Fas/CD95. We also found that although hESC and hiPSC contain all the proteins required for efficient induction and progression of extrinsic apoptotic signaling, they are resistant to TRAIL-induced apoptosis. However, both hESC and hiPSC can be sensitized to TRAIL-induced apoptosis by co-treatment with protein synthesis inhibitors such as the anti-leukemia drug homoharringtonine (HHT). HHT treatment led to suppression of cellular FLICE inhibitory protein (cFLIP) and Mcl-1 expression and, in combination with TRAIL, enhanced processing of caspase-8 and full activation of caspase-3. cFLIP likely represents an important regulatory node, as its shRNA-mediated down-regulation significantly sensitized hESC to TRAIL-induced apoptosis. Thus, we provide the first evidence that, irrespective of their origin, human pluripotent stem cells express canonical components of the extrinsic apoptotic system and on stress can activate death receptor-mediated apoptosis.
- MeSH
- antigeny CD95 genetika metabolismus MeSH
- apoptóza účinky léků MeSH
- buněčná diferenciace MeSH
- embryonální kmenové buňky cytologie účinky léků metabolismus MeSH
- FLIP (buněčný) antagonisté a inhibitory genetika metabolismus MeSH
- harringtoniny farmakologie MeSH
- inhibitory syntézy proteinů farmakologie MeSH
- kaspasa 3 genetika metabolismus MeSH
- kaspasa 8 genetika metabolismus MeSH
- lidé MeSH
- malá interferující RNA genetika metabolismus MeSH
- pluripotentní kmenové buňky cytologie účinky léků metabolismus MeSH
- proliferace buněk MeSH
- protein MCL-1 genetika metabolismus MeSH
- protein TRAIL genetika metabolismus farmakologie MeSH
- receptory TNF - typ I genetika metabolismus MeSH
- regulace genové exprese MeSH
- signální transdukce MeSH
- synergismus léků MeSH
- TRAIL receptory genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) can selectively trigger apoptosis in various cancer cell types. However, many cancer cells are resistant to death receptor-mediated apoptosis. Combination therapy with platinum complexes may affect TRAIL-induced signaling via modulation of various steps in apoptotic pathways. Here, we show that cisplatin or a more potent platinum(IV) complex LA-12 used in 20-fold lower concentration enhanced killing effects of TRAIL in human colon and prostate cancer cell lines via stimulation of caspase activity and overall apoptosis. Both platinum complexes increased DR5 surface expression in colon cancer cells. Small interfering RNA-mediated DR5 silencing rescued cells from sensitizing effects of platinum drugs on TRAIL-induced caspase-8 activation and apoptosis, showing the functional importance of DR5 in the effects observed. In addition, both cisplatin and LA-12 triggered the relocalization of DR4 and DR5 receptors to lipid rafts and accelerated internalization of TRAIL, which may also affect TRAIL signaling. Collectively, modulations of the initial steps of the extrinsic apoptotic pathway at the level of DR5 and plasma membrane are important for sensitization of colon and prostate cancer cells to TRAIL-induced apoptosis mediated by LA-12 and cisplatin.
- MeSH
- amantadin * analogy a deriváty farmakologie MeSH
- apoptóza * fyziologie účinky léků MeSH
- cisplatina * farmakologie MeSH
- fluorescenční protilátková technika MeSH
- konfokální mikroskopie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádory * metabolismus MeSH
- organoplatinové sloučeniny * farmakologie MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- protein TRAIL * metabolismus MeSH
- průtoková cytometrie MeSH
- RNA interference MeSH
- separace buněk MeSH
- signální transdukce fyziologie účinky léků MeSH
- TRAIL receptory metabolismus MeSH
- transport proteinů účinky léků MeSH
- western blotting MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
TRAIL, a ligand of the TNFalpha family, induces upon binding to its pro-death receptors TRAIL-R1/DR4 and TRAIL-R2/DR5 the apoptosis of cancer cells. Activated receptors incite the formation of the Death-Inducing Signaling Complex followed by the activation of the downstream apoptotic signaling. TRAIL-induced apoptosis is regulated at multiple levels, one of them being the presence and relative number of TRAIL pro- and anti-apoptotic receptors on the cytoplasmic membrane. In a yeast two-hybrid search for proteins that interact with the intracellular part (ICP) of DR4, we picked ARAP1, an adapter protein with ArfGAP and RhoGAP activities. In yeast, DR4(ICP) interacts with the alternatively spliced ARAP1 lacking 11 amino acids from the PH5 domain. Transfected ARAP1 co-precipitates with DR4 and co-localizes with it in the endoplasmic reticulum/Golgi, at the cytoplasmic membrane and in early endosomes of TRAIL-treated cells. ARAP1 knockdown significantly compromises the localization of DR4 at the cell surface of several tumor cell lines and slows down their TRAIL-induced death. ARAP1 overexpressed in HEL cells does not affect their TRAIL-induced apoptosis or the membrane localization of DR4, but it enhances the cell-surface presentation of phosphatidyl serine. Our data indicate that ARAP1 is likely involved in the regulation of the cell-specific trafficking of DR4 and might thus affect the efficacy of TRAIL-induced apoptosis.
- MeSH
- apoptóza účinky léků MeSH
- buněčná membrána metabolismus MeSH
- buněčné linie MeSH
- down regulace MeSH
- financování organizované MeSH
- lidé MeSH
- malá interferující RNA farmakologie MeSH
- mapování interakce mezi proteiny MeSH
- nádorové buněčné linie MeSH
- protein TRAIL metabolismus MeSH
- proteiny aktivující GTPasu fyziologie MeSH
- receptory TNF metabolismus MeSH
- techniky dvojhybridového systému MeSH
- transport proteinů fyziologie MeSH
- transportní proteiny fyziologie MeSH
- Check Tag
- lidé MeSH