Apoptotic Tumor Cell Death Research Articles

Minit-Livin Conjugated to Targeted Nanoparticles Inhibits Tumorigenicity

We found that Livin, an Inhibitor of Apoptosis Protein, is specifically cleaved to produce a truncated Livin protein (tLivin) and demonstrated the paradoxical pro-apoptotic activity of tLivin. mini-tLivin (mtLivin) is a 70 aa derivative of tLivin that remarkably, exhibits a pro-apoptotic activity as potent as tLivin. Our findings regarding tLivin and mtLivin suggest unique therapeutic avenue against cancer. To improve the delivery and efficacy of mtLivin for treatment we conjugated mtLivin-to nanoparticles (NPs) of poly (lactide-co-glycolide) (PLGA) and targeted the nano-delivery system to diffuse large B-cell lymphoma (DLBCL) using CD40L. Delivery of mtLivin with nanoparticles should enhance stability, provide prolonged bioavailability, intracellular uptake and efficacy.We generated bifunctional mtLivin-CD40L-NPs to target mtLivin-NPs to DLBCL cells. Various cell lines were used to evaluate the biological activity of free and conjugated (targeted or not) mtLivin including 293T, 721.221 B cells, L428 Hodgkin's lymphoma, OCI-LY19 DLBCL cells and OCI-LY19- GLuc cells that constitutively express a gaussia luciferase reporter.Targeted, bifunctional mtLivin-CD40L-NPs elicited significant cell death of DLBCL cells while monofunctional mtLivin-NPs had a lower effect. mtLivin-NPs may induce cell death of some cell lines without targeting (e.g. 293T) yet targeting is required to induce marked cell death of DLBCL cells.To best mimic lymphoma disease in humans, a disseminated lymphoma model was used to evaluate the antitumor effect of targeted mtLivin treatment. NOD/SCID mice were injected with OCI-LY19- GLuc cells or into the tail vein. Gaussia luciferase reporter allows IVIS imaging to monitor tumor size and location in mice. Mice were treated approximately once a week with various mtLivin-NPs formulations. Luciferase signal was observed as early as day 2 after tumor cells injection.Immunohistochemistry for anti-CD20, a human B-cell marker, showed infiltration of OCI-ly19-Gluc cells in spleen and brain close to tumor cells injection (Day 2). With time, lymphoma infiltrations were detected in brain, bone marrow, lungs, spleen and liver. All control mice exhibited paralysis and did not survive beyond 28 days. In contrast, 37.5% of animals receiving mtLivin-NPs and 71.4 % of animals receiving mtLivin-CD40L-NPs achieved complete pathological tumor response and survived significantly longer. All treatments in this study were well-tolerated.In terms of mechanism, we show high caspase-3 activity in tumors from mice that were treated with mtLivin-CD40L-NPs demonstrating the ability of the nanoparticles to target the tumors and to induce apoptotic tumor cell death. The resistance of tumor cells to drug-induced apoptosis is a major cause of cancer treatment failure. We designed mtLitvin targeted nanoparticles constituting novel personalized therapeutic approach for resistant cancer. DisclosuresNo relevant conflicts of interest to declare.

Thymoquinone‐induced antitumor and apoptosis in human lung adenocarcinoma cells

Lung cancer has been associated with the highest cancer-associated mortality rate in the world. Chemotherapeutic management of cancer necessitates introducing new promising agents. Plants represent a rich source of new antineoplastic and chemotherapeutic agents. Thymoquinone (TQ), the main constituent of Nigella sativa (black seed or black cumin), has shown potent antioxidant and anti-inflammatory activities so far. The purpose of the current study was to evaluate the antineoplastic potential of TQ and their underlying mechanisms in A549 cells (human lung cancer cell line). The A549 cells were treated with the different concentrations of TQ for three following days. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Necrosis and apoptosis were assessed by fluorescence-activated cell sorter analysis through propidium iodide and annexin V staining and also by assessing caspase-3 and -9 activation. DNA fragmentation was monitored by gel electrophoresis. TQ decreased the viability and increased apoptotic cell death in A549 human lung tumor cells. TQ treatment significantly elevated the Bax/ Bcl-2 ratio in the lung cancer cells. TQ also upregulated p53 expression, another apoptotic modulator in A549 cancer cells. TQ also activated caspase-dependent apoptosis by the activation of caspases-3 and -9. Our results proposed that TQ may be a potential new therapeutic agent for the management of lung cancer. TQ promoted apoptosis in A546 lung cancer cells by the activation of p53 and caspase cascade dependent pathways.

Abstract 4850: The minor groove binding agent quinacrine inhibits growth and increases apoptotic death in diffuse intrinsic pontine glioma tumor cells

Abstract Diffuse intrinsic pontine glioma (DIPG) is an invasive, incurable and aggressive pediatric brain tumor found in the brainstem. Improved understanding of the biology of DIPG tumors is urgently needed to develop novel treatments. Our previous studies have shown that DIPG tumors and cell lines express high levels of the DNA-binding stem cell factor high mobility group AT-hook 2 (HMGA2).. Targeting HMGA2 using lentiviral shRNA decreased DIPG cell invasion, proliferation and increased apoptosis in our studies. We hypothesized that inhibiting HMGA2 using DNA minor groove binding drugs like quinacrine would decrease DIPG proliferation and increase apoptotic cell death. We used three DIPG cell lines (JHHDIPG1, JHHDIPG16A, and SUDIPG13) to test the effect of quinacrine. Quinacrine has traditionally been used as an anti-malarial drug and is known to strongly bind to DNA. We used BrdU incorporation as a measure of proliferation and expression of cleaved caspase-3 (CC-3) as a measure of apoptosis. Treatment of DIPG cells with quinacrine showed a dose dependent increase in cell death (CC-3 expression) from 3uM to 20uM, with the most effective doses being from 3uM to 5uM. In all three DIPG cell lines, treatment with 1uM to 5uM quinacrine showed a significant reduction in cell proliferation (BrdU) from 3uM to 5uM compared to vehicle-treated cells (P<0.0001). Additionally, treatment of DIPG cell lines with 1uM to 5uM quinacrine showed a significant increase in apoptotic cell death (CC-3 expression) compared to vehicle control cells from 3uM to 5uM (P<0.001). Our data suggests that minor groove binding agents like quinacrine could be effective therapies for this lethal brain tumor. Our future studies are aimed at testing the efficacy of quinacrine in inhibiting DIPG tumorigenicity and elucidating the mechanism of action. Citation Format: Huizi Guo, Harpreet Kaur, Sepehr Akhtarkhavari, Charles G. Eberhart, Eric H. Raabe. The minor groove binding agent quinacrine inhibits growth and increases apoptotic death in diffuse intrinsic pontine glioma tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4850.

Inhibition of Carbonic Anhydrase IX by Ureidosulfonamide Inhibitor U104 Reduces Prostate Cancer Cell Growth, But Does Not Modulate Daunorubicin or Cisplatin Cytotoxicity.

Carbonic anhydrase (CA) IX has emerged as a promising target for cancer therapy. It is highly upregulated in hypoxic regions and mediates pH regulation critical for tumor cell survival as well as extracellular acidification of the tumor microenvironment, which promotes tumor aggressiveness via various mechanisms, such as augmenting metastatic potential. Therefore, the aim of this study was to analyze the complex interdependency between CA IX and the tumor microenvironment in prostate tumor cells with regard to potential therapeutic implications. CA IX was upregulated by hypoxia as well as acidosis in prostate cancer cells. This induction did not modulate intracellular pH but led to extracellular acidification. Pharmacological inhibition of CA IX activity by U104 (SLC-0111) resulted in a reduction in tumor cell growth and an increase in apoptotic cell death. Intracellular pH was reduced under normoxic and even more so under hypoxic conditions when CA IX level was high. However, although intracellular pH regulation was disturbed, targeting CA IX in combination with daunorubicin or cisplatin did not intensify apoptotic tumor cell death. Hence, targeting CA IX in prostate cancer cells can lead to intracellular pH dysregulation and, consequently, can reduce cellular growth and elevate apoptotic cell death. Attenuation of extracellular acidification by blocking CA IX might additionally impede tumor progression and metastasis. However, no beneficial effect was seen when targeting CA IX in combination with chemotherapeutic drugs.

LQFM030 reduced Ehrlich ascites tumor cell proliferation and VEGF levels

AimsThis study reports the biological properties of LQFM030 in vivo, a molecular simplification of the compound nutlin-1. Main methodsEhrlich ascites tumor (EAT)-bearing mice were treated intraperitoneally with LQFM030 (50, 75 or 150mg/kg) for 10days to determine changes in ascites tumor volume, body weight, cytotoxicity and angiogenesis. Moreover, flow cytometric expression of p53 and p21 proteins and caspase-3/7, −8 and −9 activation were investigated in EAT cells from mice treated. Acute oral systemic toxicity potential of LQFM030 in mice was also investigated using an alternative method. Key findingsTreatment of EAT-bearing mice with LQFM030 resulted in a marked decline in tumor cell proliferation and the vascular endothelial growth factor (VEGF) levels along with enhanced survival of the mice. Apoptotic tumor cell death was detected through p53 and p21 modulation and increase of caspase-3/7, −8 and −9 activity. LQFM030 also showed orally well tolerated, being classified in the UN GHS category 5 (LD50>2000–5000mg/Kg). SignificanceLQFM030 seems to be a promising antitumor candidate for combinatory therapy with typical cytotoxic compounds, reducing the toxicity burden while allowing a superior anticancer activity. Moreover, these data also open new perspectives for LQFM030 as an antiangiogenic agent for treatment of diseases involving VEGF overexpression.

In vivo knockdown of antisense non‐coding mitochondrial RNAs by a lentiviral‐encoded shRNA inhibits melanoma tumor growth and lung colonization

The family of non-coding mitochondrial RNAs (ncmtRNA) is differentially expressed according to proliferative status. Normal proliferating cells express sense (SncmtRNA) and antisense ncmtRNAs (ASncmtRNAs), whereas tumor cells express SncmtRNA and downregulate ASncmtRNAs. Knockdown of ASncmtRNAs with oligonucleotides induces apoptotic cell death of tumor cells, leaving normal cells unaffected, suggesting a potential application for developing a novel cancer therapy. In this study, we knocked down the ASncmtRNAs in melanoma cell lines with a lentiviral-encoded shRNA approach. Transduction with lentiviral constructs targeted to the ASncmtRNAs induced apoptosis in murine B16F10 and human A375 melanoma cells in vitro and significantly retarded B16F10 primary tumor growth in vivo. Moreover, the treatment drastically reduced the number of lung metastatic foci in a tail vein injection assay, compared to controls. These results provide additional proof of concept to the knockdown of ncmtRNAs for cancer therapy and validate lentiviral-shRNA vectors for gene therapy.

Antitumor activity of gemcitabine against high-grade meningioma in vitro and in vivo.

Currently, there is no established therapeutic option for high-grade meningioma recurring after surgery and radiotherapy, and few chemotherapeutic agents are in development for the treatment of high-grade meningioma. Here in this study, we screened a panel of chemotherapeutic agents for their possible antitumor activity in high-grade meningioma and discovered that high-grade meningioma cells show a preferential sensitivity to antimetabolites, in particular, to gemcitabine. In vitro, gemcitabine inhibited the growth of high-grade meningioma cells effectively by inducing S-phase arrest and apoptotic cell death. In vivo, systemic gemcitabine chemotherapy suppressed not only tumor initiation but also inhibited the growth and achieved a long-term control of established tumors in xenograft models of high-grade meningioma. Histological analysis indicated that systemic gemcitabine blocks cell cycle progression and promotes apoptotic cell death in tumor cells in vivo. Together, our data demonstrate that gemcitabine exerts potent antitumor activity against high-grade meningioma through cytostatic and cytotoxic mechanisms. We therefore propose gemcitabine is a promising chemotherapeutic agent that warrants further investigation as a treatment option for high-grade meningioma.

Abstract AP26: SMAC–MIMETIC TREATMENT ENHANCES ANTITUMOR EFFECT OF STANDARD CHEMOTHERAPY IN OVARIAN CANCERS EXPRESSING LOW CASPASE 8 IN VITRO AND IN VIVO

Abstract Ovarian cancer is the most lethal gynecological cancer in the US. Standard treatment consists of surgery followed by chemotherapies relying on apoptotic tumor cell death. Most women with advanced stage disease will relapse, suggesting that this disease is characterized by primary and acquired resistance to chemotherapy, and targeted approaches to treatment are greatly needed. We recently demonstrated that low Caspase 8 expression levels in ovarian cancers correlated with resistance to chemotherapy, and a subpopulation of ovarian cancer patients with low Caspase 8 levels exhibits poorer overall survival after standard of care treatment. We hypothesized that the loss of Caspase 8 function reduces the cancer cells' ability to undergo apoptosis when exposed to standard chemotherapy. Here we show that combination treatment with Smac-mimetics targets tumor cells with low Caspase 8 and induces necroptotic cell death. We investigated the in vitro sequential effect of Smac-mimetic added to carboplatin + paclitaxel treatment of ovarian cancer cells expressing wild type, low and mutant Caspase 8 levels, which resulted in a 2-4fold enhancement of cell death. In vivo treatment of mice bearing subcutaneous or intraperitoneal ovarian xenografts showed greater aggressiveness of Caspase 8 deficient vs wild-type tumors; combined treatment with chemotherapy and Smac-mimetic resulted in over 50% decrease in low-Caspase 8 xenograft growth, as well as enhanced overall survival of the mice. Subpopulations of women with low Caspase 8 ovarian cancers that resist apoptosis could benefit from treatments which promote non-apoptotic forms of cell death, such as Smac-mimetic induced necroptosis. Clinical validation of such approaches are needed to increase the effectiveness of current standard ovarian cancer treatment. Citation Format: Lídia Hernandez and Christina M. Annunziata. SMAC–MIMETIC TREATMENT ENHANCES ANTITUMOR EFFECT OF STANDARD CHEMOTHERAPY IN OVARIAN CANCERS EXPRESSING LOW CASPASE 8 IN VITRO AND IN VIVO [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP26.

Effect of ONC201 and bortezomib on apoptosis in non-Hodgkin'™s lymphoma (NHL) xenografts.

e19016 Background: Our group has previously shown preclinical in vitro efficacy of novel small molecule, ONC201 across several pediatric NHL cell lines (Talekar et al, 2015) as a monoagent as well as in combination with chemotherapy agents, significantly cytarabine and bortezomib. We investigated the preclinical efficacy of ONC201 and bortezomib combination in vivo in a NHL mouse xenograft model. Methods: Burkitt’s lymphoma cell line (Ramos) transduced with lentiviral GFP was utilized to establish subcutaneous flank xenografts in 5-7 week(wk) old immunodeficient SCID mice [NOD.Cg- Prkdcscid Il2rgtm1Wjl/SzJ]. All tumors were established by Day 10, detected by bioluminescent imaging and were serially imaged x1/wk. Mice were studied in 4 treatment arms (7 mice per arm) as tabulated in the table. Results: Mice in Arms 1, 2 & 3 had continued tumor growth and were humanely euthanized during Wk 3- 4 of treatment when the tumor volume reached the maximum allowable size per the approved institutional IACUC protocol. Mice in Arm 4 showed gross tumor necrosis and had to be euthanized in Wk 4. Upon treatment initiation, 2 mice in each arm were sacrificed at 48 and 72 hours respectively and tumor samples harvested for histology and IHC analysis (TRAIL and CHOP/GADD). Histological analysis revealed that the control group had predominantly viable tumor cells at both 48 & 72 H. The monoagent groups showed minimal cell death with bortezomib alone and modest cell death with ONC201 as monoagent in clustered regions. In contrast, the combination group showed: i) extensive global tumor necrosis with nuclear debris on H&E, ii) prominent TRAIL induction by IHC analysis, and, iii) significant apoptotic cell death accompanied by CHOP /GADD overexpression indicating potential ER stress. Induction of these pharmacodynamic markers in Arm 4 were evident at 48H and more prominent at the 72H. Conclusions: ONC201 effectively synergizes with bortezomib to cause apoptotic tumor cell death in pediatric NHL murine xenograft and merits further investigation. [Table: see text]

Phytochemical Inhibition of Multidrug Resistance Protein-1 as a Therapeutic Strategy for Hemangioendothelioma.

Hemangiomas are endothelial cell tumors and the most common soft tissue tumors in infants. They frequently cause deformity and can cause death. Current pharmacologic therapies have high-risk side-effect profiles, which limit the number of children who receive treatment. The objectives of this work were to identify the mechanisms through which standardized berry extracts can inhibit endothelial cell tumor growth and test these findings in vivo. EOMA cells are a validated model that generates endothelial cell tumors when injected subcutaneously into syngeneic (129P/3) mice. EOMA cells treated with a blend of powdered natural berry extracts (NBE) significantly inhibited activity of multidrug resistance protein-1 (MRP-1) compared to vehicle controls. This resulted in nuclear accumulation of oxidized glutathione (GSSG) and apoptotic EOMA cell death. When NBE-treated EOMA cells were injected into mice, they generated smaller tumors and had a higher incidence of apoptotic cell death compared to vehicle-treated EOMA cells as demonstrated by immunocytochemistry. Kaplan-Meier survival curves for tumor-bearing mice showed that NBE treatment significantly prolonged survival compared to vehicle-treated controls. These are the first reported results to show that berry extracts can inhibit MRP-1 function that causes apoptotic tumor cell death by accumulation of GSSG in the nucleus of EOMA cells where NADPH oxidase is hyperactive and causes pathological angiogenesis. These findings indicate that berry extract inhibition of MRP-1 merits consideration and further investigation as a therapeutic intervention and may have application for other cancers with elevated MRP-1 activity. Antioxid. Redox Signal. 26, 1009-1019.

Selection of antitumor displayed peptides for the specific delivery of the anticancer drug lactaptin.

It has been previously demonstrated that lactaptin, the proteolytic fragment of human milk protein κ-casein, induces the death of various cultured cancer cells. The recombinant analog of lactaptin, RL2, effectively induces the apoptosis of mouse hepatocarcinoma-1 (HA-1) tumor cells in vitro and suppress the growth of HA-1 tumors and metastases in vivo. The antitumor drug Lactaptin developed on the basis of RL2 has been successful in preclinical trials. Lactaptin shows its efficiency in relation to mouse and human cancer cells and tumors. However, Lactaptin, as with the majority of protein-based therapeutic drugs, is distributed evenly throughout the organism, which reduces its antitumor efficacy. To develop the targeted delivery of lactaptin, the present study selected tumor-specific peptides by screening a phage display peptide library in vivo on A/Sn strain mice with subcutaneously transplanted HA-1 cells. Two genetic constructs were made for the production of recombinant fusion proteins composed of RL2 and the selected tumor-targeting peptide. In vitro experiments involving HA-1, MDA-MB-231 and MCF-7 cells cultures demonstrated that the fusion proteins induce apoptotic death in mouse and human tumor cells, as with RL2. The in vivo experiments involving the mouse HA-1 tumor model demonstrated that the tumor fluorescence intensity of the Cy5-fusion protein conjugates is higher than that of RL2-Cy5. As conjugation of the tumor-specific peptides to RL2 provided retention of RL2 in the tumor tissues, fusion proteins composed of lactaptin and peptides specific for human tumors are deemed promising to improve the antitumor efficiency of lactaptin.

The in vivo antitumor effect of the apoptin-producing recombinant vaccinia virus strain is associated with blockage of mitotic division of cancer cells

Vaccinia virus (VACV) possesses a natural oncolytic activity, for the enhancement of which genes encoding various effector molecules, for example, those inducing apoptotic death of tumor cells, are introduced into the virus genome. One such transgene is the gene encoding apoptin protein. The aim of the current work was to study antitumor activity of the apoptin-producing recombinant VACV strain VVdGF-ApoS24/2 in a syngenic murine tumor model. An Ehrlich carcinoma was implanted subcutaneously or intraperitoneally into C57B1 line mice. After tumor development, mice were intratumorally injected with 107 PFU/mouse of VVdGF-ApoS24/2 virus in a single dose, while control mice received 0.9% NaCl solution. Animals were sacrificed at different times after VACV injection. Tumor samples and ascetic fluid cells were fixed in 4% paraformaldehyde and further studied by light microscopy, immunohistochemistry, and electron microscopy. Virus titers in tumor cells were determined by the PFU method. VVdGF-ApoS24/2 VACV strain caused a significant reduction in the volumes of both solid and ascites Ehrlich carcinomas compared to the tumors in the control group of mice, although the virus reproduction rate in the tumor cells was rather low. The antitumor effect of VVdGF-ApoS24/2 could neither be attributed to virus-induced destruction, necrosis, or apoptosis of tumor cells nor to accumulation of the immune-effector cells in the tumors. A decrease in the number of tumor cells undergoing mitosis was observed when examining carcinoma sections, while counting Ki-67 and PCNA positive cells and analysis of their numbers showed that VVdGF-ApoS24/2 strain arrests cell cycle in the S-phase, thereby blocking tumor-cell division and slowing down tumor growth. The obtained results indicate that the insertion of the gene encoding apoptin into the genome of the original L-IVP VACV strain improved the ability of the virus to arrest the cell cycle in Ehrlich carcinoma cells.

SU‐F‐J‐225: Histology Study of MR Guided Pulsed Focused Ultrasound On Treatment of Prostate Cancer in Vivo

Purpose:Our previous study demonstrated significant tumor growth delay in the mice treated with pulsed high intensity focused ultrasound (pHIFU). The purpose of this study is to understand the cell killing mechanisms of pHIFU.Methods:Prostate cancer cells (LNCaP), were grown orthotopically in 17 nude mice. Tumor‐bearing mice were treated using pHIFU with an acoustic power of 25W, pulse width 100msec and 300 pulses in one sonication under MR guidance. Mutiple sonications were used to cover the whole tumor volume. The temperature (less than 40 degree centigrade in the focal spot) was monitored using MR thermometry. Animals were euthanized at pre‐determined time points (n=2) after treatment: 0 hours; 6 hrs; 24 hrs; 48 hrs; 4 days and 7 days. Two tumorbearing mice were used as control. Three tumor‐bearing mice were treated with radiation (RT, 2 Gy) using 6 MV photon beams. RT treated mice were euthanized at 0 hr, 6 hrs and 24 hrs. The tumors were processed for immunohistochemical (IHC) staining for PARP (a surrogate of apoptosis). A multispectral imaging analysis system was used to quantify the expression of PARP staining. Cell apoptosis was calculated based on the PARP expression level using the DAB analysis software.Results:Our data showed that PARP related apoptosis peaked at 48 hrs and 7 days in pHIFU treated mice, which is comparable to that for the RT group at 24 hrs. The preliminary results from this study were consistent with our previous study on tumor growth delay using pHIFU.Conclusion:Our results demonstrated that non‐thermal pHIFU increased apoptotic tumor cell death through the PARP related pathway. MR guided pHIFU may have a great potential as a safe, noninvasive treatment modality for cancer therapy. This treatment modality may synergize with PARP inhibitors to achieve better therapeutic result.

The Tag7-Hsp70 cytotoxic complex induces tumor cell necroptosis via permeabilisation of lysosomes and mitochondria.

Tag7 (PGRP-S, or PGLYRP1), an innate immunity protein, plays an important role in the immune defense system. It forms a stable cytotoxic complex with the heat shock protein Hsp70. This complex can induce an apoptotic or necroptotic tumor cell death by interacting with the TNFR1 receptor. In this study, we analyzed molecular events involved in the process of the Tag7-Hsp70-induced necroptosis. We found that Tag7 can bind to sTNFR1, a soluble fragment of the TNFR1 receptor, leading to an inhibition of the RIP1 dependent necroptosis. A major role in the downstream phases of the Tag7-Hsp70 induced necroptosis was played by an interaction between lysosomes and mitochondria. The interaction of Tag7-Hsp70 with the TNFR1 receptor triggered a certain sequence of events: at first, it activated RIP1 kinase, and later on, increased intracellular concentration of Са(2+) ions and an activation of calpains, which led to the permeabilization of the lysosomal membranes. The consequent release of the lysosomal enzymes, including cathepsins B and D, resulted in the depolarization of the mitochondrial membrane, ROS production, and eventual cell death.

Cell survival of glioblastoma grown in medium containing hydrogen peroxide and/or nitrite, or in plasma-activated medium

Non-equilibrium atmospheric pressure plasmas generate a high electron density (on the order of 1016 electrons per cm−3) using Ar gas. Culture medium in air at room temperature was plasma-irradiated for several hundred seconds. Tens of micromolar hydrogen peroxide (H2O2) and millimolar levels of nitrous ion (NO2−) were detected in the plasma-irradiated culture medium (plasma activated medium; PAM) and selectively induced the apoptotic death of glioblastoma tumor cells, but did not kill normal mammary epithelial cells. A similar antitumor effect was induced by spiking the medium with comparable concentrations of H2O2 and NO2−. The PAM remained still a somewhat difference that it should also be assessed for understanding other latent mechanisms.

Apoptotic tumor cell death under the influence of the cytotoxic complex Tag7-Hsp70 is induced by interaction with the TNFR1 receptor.

The results of comparative analysis of interaction between the protein cytotoxic complex Tag 7-Hsp70 and the Tag 7 component of this complex with TNFR1 receptor in solution and in tumor cells are presented.

Macrophage Polarization In The Tumor Microenvironment

Tumor associated macrophages (TAMs) are known to support tumor progression and their accumulation is generally associated with poor prognosis. The shift from a tumor-attacking to a tumor-supportive macrophage phenotype is based on an educational program that, at least in part, is initiated by apoptotic tumor cells. We explored the macrophage phenotype shift during tumor progression by analyzing the macrophage NO-output system and examining potential NO targets. Biochemical and Molecular Biology-orientated cell culture experiments, in part using 3d-tumor spheroid models as well as animal experiments were used. Apoptotic cells polarize macrophages towards a healing, tumor-supportive phenotype. Soluble mediators released from apoptotic cells, among them the lipid sphingosine-1-phosphate (S1P), cause expression of arginase 2 in macrophages, thereby lowering citrulline/NO formation but enhancing ornithine production. Mechanistically, this is achieved via the S1P2 receptor and the CRE (cAMP-response element) binding site in the arginase 2 promoter. Reduced NO-formation is also seen in ex vivo macrophages from a xenograft model allowing restricted vs. unrestricted tumor growth based on tumor-associated S1P-formation. The theoretical ability of NO to target hypoxia-inducible factor-1 (HIF-1) and jumonji histone demethylases (JHDMs) in cells of the tumor microenvironment will be discussed in light of the iNOS/arginase balance. Moreover, data on the importance of HIF-1 in macrophages for their interaction with tumor cells, polarization, and angiogenic potential will be presented. We hypothesize that apoptotic death of tumor cells and associated macrophage activation facilitates the progression of malignant disease. The macrophage polarization program affects the NO-output system and the capacity of macrophages to support or restrict tumor growth.

The pitfall of the transient, inconsistent anticancer capacity of antiestrogens and the mechanism of apparent antiestrogen resistance.

Although antiestrogens have been available for breast cancer therapy since the early 1970s, neither their inconsistent anticancer capacity nor the developing antiestrogen resistance of tumors can be fully understood. Although clinical and experimental investigations revealed many tiny details concerning the link between estrogen signaling and tumor development, they yielded fairly controversial findings. Estrogen receptor (ER) overexpression in tumor cells induced by estrogen treatment was erroneously regarded as a promoter of DNA damage, genomic instability, and tumor growth. Similarly, compensatory ER overexpression caused by antiestrogen treatment or estrogen withdrawal was mistakenly evaluated as a key for rapid tumor growth attributed to acquired antiestrogen resistance. Nevertheless, ER upregulation induced by estrogen treatment is a physiologic process even in tumor cells, whereas in the case of antiestrogen administration, it is a contraregulatory action to defend the endangered estrogen signaling. Upregulation of estrogen signaling displays a unique dichotomy, ensuring the survival and safe proliferative activity of healthy cells, while inducing apoptotic death of malignant tumor cells. Analysis of the fairly controversial results justifies that whatever type of available endocrine therapies may be used, including estrogen, antiestrogen treatment, or oophorectomy, an extreme upregulation of ER signaling seems to be the crucial mechanism of successful prevention and treatment for breast cancer. The inconsistent therapeutic effects of antiestrogen administration may be explained by the different genetic capacities of patients for the compensatory upregulation of ER and aromatase enzyme expressions. The weaker the defensive counteraction against the inhibition of estrogen signaling, the poorer is the prognosis of the disease. De novo or acquired antiestrogen resistance of tumors may be associated with the missing capacity of patients for the extreme upregulation of estrogen signaling or with the exhaustion of defensive counteractions in cases that previously showed good reactivity. High-dose estrogen treatment is capable of restoring ER signaling and anticancer capacity even after heavy exposure to antiestrogen therapy.

Targeting TNF-related apoptosis-inducing ligand (TRAIL) receptor by natural products as a potential therapeutic approach for cancer therapy.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been shown to selectively induce apoptotic cell death in various tumor cells by engaging its death-inducing receptors (TRAIL-R1 and TRAIL-R2). This property has led to the development of a number of TRAIL-receptor agonists such as the soluble recombinant TRAIL and agonistic antibodies, which have shown promising anticancer activity in preclinical studies. However, besides activating caspase-dependent apoptosis in several cancer cells, TRAIL may also activate nonapoptotic signal transduction pathways such as nuclear factor-kappa B, mitogen-activated protein kinases, AKT, and signal transducers and activators of transcription 3, which may contribute to TRAIL resistance that is being now frequently encountered in various cancers. TRAIL resistance can be overcome by the application of efficient TRAIL-sensitizing pharmacological agents. Natural compounds have shown a great potential in sensitizing cells to TRAIL treatment through suppression of distinct survival pathways. In this review, we have summarized both apoptotic and nonapoptotic pathways activated by TRAIL, as well as recent advances in developing TRAIL-receptor agonists for cancer therapy. We also briefly discuss combination therapies that have shown great potential in overcoming TRAIL resistance in various tumors.

Combined Treatment of Herbal Mixture Extract H9 with Trastuzumab Enhances Anti-tumor Growth Effect

Extracts from Asian medicinal herbs are known to be successful therapeutic agents against cancer. In this study, the effects of three types of herbal extracts on anti-tumor growth were examined. Among the three types of herbal extracts, H9 showed stronger anti-tumor growth effects than H5 and H11 in vivo. To find the molecular mechanism by which H9 inhibited the proliferation of breast cancer cell lines, the levels of apoptotic markers were examined. Proapoptotic markers, including cleaved PARP and cleaved caspases 3 and 9, were increased, whereas the anti-apoptotic marker Bcl-2 was decreased by H9 treatment. Next, the combined effect of H9 with the chemotherapeutic drugs doxorubicin/cyclophosphamide (AC) on tumor growth was examined using 4T1-tumor-bearing mice. The combined treatment of H9 with AC did not show additive or synergetic anti-tumor growth effects. However, when tumor-bearing mice were co-treated with H9 and the targeted anti-tumor drug trastuzumab, a delay in tumor growth was observed. The combined treatment of H9 and trastuzumab caused an increase of natural killer (NK) cells and a decrease of myeloid-derived suppressor cells (MDSC). Taken together, H9 induces the apoptotic death of tumor cells while increasing anti-tumor immune activity through the enhancement of NK activity and diminishment of MDSC.