Pro-apoptotic Genes Puma Research Articles

Synergistic Enhancement of Carboplatin Efficacy through pH-Sensitive Nanoparticles Formulated Using Naturally Derived Boswellia Extract for Colorectal Cancer Therapy.

Carboplatin (Cp) is a potent chemotherapeutic agent, but its effectiveness is constrained by its associated side effects. Frankincense, an oleo-gum resin from the Boswellia sacra tree, has demonstrated cytotoxic activity against cancer cells. This study explored the synergistic potential of nanoparticles formulated from Boswellia sacra methanolic extract (BME), to enhance the therapeutic efficacy of Cp at reduced doses. Nanoparticles were prepared via the nanoprecipitation method, loaded with Cp, and coated with positively charged chitosan (CS) for enhanced cell interaction, yielding Cp@CS/BME NPs with an average size of 160.2 ± 4.6 nm and a zeta potential of 12.7 ± 1.5 mV. In vitro release studies revealed a pH-sensitive release profile, with higher release rates at pH 5.4 than at pH 7.4, highlighting the potential for targeted drug delivery in acidic tumor environments. In vitro studies on HT-29 and Caco-2 colorectal cancer cell lines demonstrated the nanoformulation's ability to significantly increase Cp uptake and cytotoxic activity. Apoptosis assays further confirmed increased induction of cell death with Cp@CS/BME NPs. Cell-cycle analysis revealed that treatment with Cp@CS/BME NPs led to a significant increase in the sub-G1 phase, indicative of enhanced apoptosis, and a marked decrease in the G1-phase population coupled with an increased G2/M-phase arrest in both cell lines. Further gene expression analysis demonstrated a substantial downregulation of the anti-apoptotic gene Bcl-2 and an upregulation of the pro-apoptotic genes Bax, PUMA, and BID following treatment with Cp@CS/BME NPs. Thus, this study presents a promising and innovative strategy for enhancing the therapeutic efficacy of chemotherapeutic agents using naturally derived ingredients while limiting the side effects.

Amygdalin as a chemoprotective agent in co-treatment with cisplatin.

Amygdalin is a naturally occurring glycoside used in traditional Chinese medicine and is known to have anti-cancer properties. Even though the anti-cancer properties of amygdalin are well known, its effect on normal cells has not been thoroughly investigated. The aim of the present study was to investigate a possible chemo-protective role of amygdalin against the cytotoxic effects of chemotherapy for normal human cells. Specifically, it was tested in combination with a strong chemotherapeutic drug cisplatin. Human non-tumorigenic MCF12F epithelial cell line, human fibroblasts cells, human breast cancer MCF7 and MDA-MB-231 cells were treated with cisplatin in a dose- and time-depended manner in the absence or presence of amygdalin. When MCF12F cells and fibroblasts underwent pre-treatment with amygdalin followed by cisplatin treatment (24 h amygdalin + 24 h cisplatin), the cell viability was increased (22%, p < 0.001) as indicated using MTT assay. As attested by flow cytometry, combination treatment was associated with decreased the percentage of late apoptotic cells compared with monotherapy (fold-change of decrease = 1.6 and 4.5 for 15 and 20 μΜ, respectively). Also, the proteins expression of PUMA, p53, phospho-p53 and Bax decreased, when a combination treatment was used vs. cisplatin alone, while the proapoptotic proteins Bcl-2 and Bcl-xL exhibited an increased tendency in the presence of amygdalin. Moreover, the levels of pro-apoptotic genes PUMA, p53, and BAX mRNA were significantly downregulated (∼83%, ∼66%, and ∼44%, respectively) vs. cisplatin alone, while the mRNA levels of anti-apoptotic genes BCl-2 and Bcl-XL were upregulated (∼44.5% and ∼51%, respectively), vs. cisplatin alone after 24 h of combination treatment. The study on the Combination index (CI) assay indicated that amygdalin could be possibly considered as an antagonist to cisplatin (2.2 and 2.3) for MCF12F and fibroblast cells, respectively. In contrast, for the breast cancer MCF7 and MDA-MB-231 cells, amygdalin and cisplatin indicated a synergistic effect (0.8 and 0.65), respectively. Our present findings suggest that amygdalin has chemo-modulatory effect when used in co-treatment with cisplatin and is able to protect normal breast cells as well as the fibroblasts during chemotherapy treatment, indicating a strong selective chemoprotective ability and may contribute to a better quality of life for cancer patients.

Loss of TAF8 causes TFIID dysfunction and p53-mediated apoptotic neuronal cell death.

Mutations in genes encoding general transcription factors cause neurological disorders. Despite clinical prominence, the consequences of defects in the basal transcription machinery during brain development are unclear. We found that loss of the TATA-box binding protein-associated factor TAF8, a component of the general transcription factor TFIID, in the developing central nervous system affected the expression of many, but notably not all genes. Taf8 deletion caused apoptosis, unexpectedly restricted to forebrain regions. Nuclear levels of the transcription factor p53 were elevated in the absence of TAF8, as were the mRNAs of the pro-apoptotic p53 target genes Noxa, Puma and Bax. The cell death in Taf8 forebrain regions was completely rescued by additional loss of p53, but Taf8 and p53 brains failed to initiate a neuronal expression program. Taf8 deletion caused aberrant transcription of promoter regions and splicing anomalies. We propose that TAF8 supports the directionality of transcription and co-transcriptional splicing, and that failure of these processes causes p53-induced apoptosis of neuronal cells in the developing mouse embryo.

Evaluation of 2-Thioxoimadazolidin-4-one Derivatives as Potent Anti-Cancer Agents through Apoptosis Induction and Antioxidant Activation: In Vitro and In Vivo Approaches.

Background: Hepatocellular carcinoma (HCC) is one of the most widespread malignancies and is reported as the fourth most prevalent cause of cancer deaths worldwide. Therefore, we aimed to investigate the probable mechanistic cytotoxic effect of the promising 2-thioxoimidazolidin-4-one derivative on liver cancer cells using in vitro and in vivo approaches. The compounds were tested for the in vitro cytotoxic activity using MTT assay, and the promising compound was tested in colony forming unit assay, flow cytometric analysis, RT-PCR, Western blotting, in vivo using SEC-carcinoma and in silico to highlight the virtual mechanism of action. Both compounds 4 and 2 performed cytotoxic effects against HepG2 cells with IC50 values of 0.017 and 0.18 μM, respectively, compared to Staurosporine and 5-Fu as reference drugs with IC50 values of 5.07 and 5.18 µM, respectively. Compound 4 treatment revealed apoptosis induction by 19.35-fold (11.42% compared to 0.59% in control), arresting the cell cycle at G2/M phase. Moreover, studying gene expression that plays critical roles in cell cycle and apoptosis by RT-PCR demonstrated that compound 4 enhances the expression of the pro-apoptotic genes p53, PUMA, and Caspase 3, 8, and 9, and impedes the anti-apoptotic Bcl-2 gene in the HepG2 cells. It can also inhibit the PI3K/AKT pathway at both gene and protein levels, which was reinforced by the in silico predictions of the molecular docking simulations towards the PI3K/AKT proteins. Finally, in vivo study verified that compound 4 has a promising anti-cancer activity through activating antioxidant levels (CAT, SOD and GSH) and ameliorating hematological, biochemical, and histopathological findings.

N-Acetyl Cysteine, Selenium, and Ascorbic Acid Rescue Diabetic Cardiac Hypertrophy via Mitochondrial-Associated Redox Regulators.

Metabolic disorders often lead to cardiac complications. Metabolic deregulations during diabetic conditions are linked to mitochondrial dysfunctions, which are the key contributing factors in cardiac hypertrophy. However, the underlying mechanisms involved in diabetes-induced cardiac hypertrophy are poorly understood. In the current study, we initially established a diabetic rat model by alloxan-administration, which was validated by peripheral glucose measurement. Diabetic rats displayed myocardial stiffness and fibrosis, changes in heart weight/body weight, heart weight/tibia length ratios, and enhanced size of myocytes, which altogether demonstrated the establishment of diabetic cardiac hypertrophy (DCH). Furthermore, we examined the expression of genes associated with mitochondrial signaling impairment. Our data show that the expression of PGC-1α, cytochrome c, MFN-2, and Drp-1 was deregulated. Mitochondrial-signaling impairment was further validated by redox-system dysregulation, which showed a significant increase in ROS and thiobarbituric acid reactive substances, both in serum and heart tissue, whereas the superoxide dismutase, catalase, and glutathione levels were decreased. Additionally, the expression levels of pro-apoptotic gene PUMA and stress marker GATA-4 genes were elevated, whereas ARC, PPARα, and Bcl-2 expression levels were decreased in the heart tissues of diabetic rats. Importantly, these alloxan-induced impairments were rescued by N-acetyl cysteine, ascorbic acid, and selenium treatment. This was demonstrated by the amelioration of myocardial stiffness, fibrosis, mitochondrial gene expression, lipid profile, restoration of myocyte size, reduced oxidative stress, and the activation of enzymes associated with antioxidant activities. Altogether, these data indicate that the improvement of mitochondrial dysfunction by protective agents such as N-acetyl cysteine, selenium, and ascorbic acid could rescue diabetes-associated cardiac complications, including DCH.

Carnosic acid exhibits antiproliferative and proapoptotic effects in tumoral NCI-H460 and nontumoral IMR-90 lung cells

ABSTRACT Carnosic acid (CA) is a phenolic diterpene with many important biological activities including antimicrobial, antioxidant, anti-inflammatory properties, and anti-proliferative properties. The aim of the present study was to investigate cytotoxic activity, cell cycle, apoptotic, and molecular effects attributed to CA in non-tumoral IMR-90 (human fetal lung fibroblasts), as well as tumoral NCI-H460 (human non–small-cell lung cancer) cell lines. Cell proliferation was evaluated by Real-Time Cell Analysis system, while apoptosis and cell cycle were assessed using flow cytometry. RT-qPCR was used to estimate the relative expression of genes involved in cell cycle regulation, DNA damage and repair, and apoptosis induction. CA inhibited proliferation of IMR-90 and NCI-H460 cells via cell cycle arrest at G0/G1 and G2/M phases, according to the treatment concentration. The mRNA levels of genes encoding cyclins A2, B1, and B2 were downregulated in response to CA treatment of IMR-90 cells. Apoptosis was induced and proapoptotic gene PUMA was upregulated in both cell lines. mRNA levels of genes ATR, CCND1, CHK1, CHK2, MYC, GADD45A, H2AFX, MTOR, TP53, and BCL2, CASP3 were not markedly changed following CA treatments. Although CA exerted antiproliferative activity against NCI-H460 tumor cells, this phytochemical induced toxic effects in non-tumoral cells, and thus needs to be considered carefully prior to pharmacological use therapeutically.

Biomarker Modulation By Mivebresib (ABBV-075) +/- Venetoclax in Relapsed/Refractory Acute Myeloid Leukemia

Biomarker Modulation By Mivebresib (ABBV-075) +/- Venetoclax in Relapsed/Refractory Acute Myeloid Leukemia

Ruthenium Complex Induce Cell Death in G-415 Gallbladder Cancer Cells.

In this work, we present a recently developed ruthenium complex that shows anticancer activity in gallbladder cancer cells. After the synthesis of the new ruthenium complexes, the antiproliferative, cytotoxicity, and apoptosis activities were evaluated in vitro by the triple assay ApoTox-Glo. Then, the transcription levels of genes related to apoptosis were evaluated by real-time PCR (q-PCR). The ruthenium complex, called Ru-UCN3, inhibits the proliferation of gallbladder cancer cells G-415 by means of apoptosis, which was demonstrated by the overexpression of the pro-apoptotic genes Puma, Diablo, and Caspasa-9 together with the repression of the anti-apoptotic genes Bcl-xL and Bcl-2. In addition, we found strong caspase 3/7 activity in the cells at 24h of the Ru-UCN3 exposure, which was evaluated by the triple ApoTox-Glo assay. The new ruthenium complexes evaluated had an inhibitory effect on G-415 cells. We think that Ru-UCN3 could be a promising anticancer agent, which should be explored with more in vitro and in vivo assays and probably with the chemical modulation of this molecule.

Abstract 2492: BRAF inhibitors synergize with BH3 mimetics to induce apoptosis in BRAF mutant colorectal cancer cells

Abstract Activating mutations in BRAF (BRAFV600E) occur in ~10% of colorectal cancers (CRCs) and drive tumorigenesis through constitutive activation of MAPK signaling. In metastatic CRC, BRAF mutations are associated with poorer prognosis and resistance to conventional therapies, necessitating an urgent need to develop new treatments for these patients. BRAF inhibitors such as vemurafenib and dabrafenib have significant clinical activity in BRAF-mutant melanoma, however BRAF-mutant CRCs are largely refractory to these agents, due at least in part to feedback-relief mediated reactivation of MAPK signaling or alternate signaling pathway activation. Strategies to enhance the activity of BRAF inhibitors in BRAF-mutant CRC are therefore needed. Consistent with clinical observations, treatment of a panel of BRAF-mutant melanoma and CRC cell lines with vemurafenib resulted in significantly increased apoptosis in melanoma cell lines compared to CRC cell lines, where effects were largely cytostatic. To determine the mechanisms for this differential response we interrogated vemurafenib-induced gene expression changes in the two tumor types, focusing on altered expression of components of the intrinsic apoptotic pathway. Vemurafenib induced a more pronounced increase in expression of the pro-apoptotic genes BIM, BMF and PUMA and suppression of pro-survival gene MCL1 in melanoma cells compared to CRC cells. These findings suggested that the extent to which expression of pro and anti-apoptotic genes are altered by vemurafenib in CRC cells may be insufficient to reach the threshold required for apoptosis initiation. We therefore postulated that BH3-mimetics may synergize with vemurafenib to induce apoptosis in BRAF-mutant CRC cells. Analysis of quantitative proteomic data of BRAF-mutant CRC cell lines revealed significantly higher basal expression of the pro-survival proteins Bcl-xL and MCL1 compared to BCL2 and BCLW, suggesting CRC cells may be particularly dependent on Bcl-xL and MCL1 for survival. Indeed, combination treatment of BRAF-mutant CRC cells with the Bcl-xL inhibitor A-1331852 significantly enhanced apoptosis in the majority of BRAF-mutant CRC lines. Comparatively, combination treatment of vemurafenib with the MCL1 inhibitor S63845 induced a modest increase in apoptosis, while combination treatment with the BCL2 inhibitor ABT-199 had no effect on apoptosis, consistent with the low levels of BCL2 expression in these lines. Finally, we investigated the effect of combination treatment of vemurafenib with inhibitors of both Bcl-xL and MCL1. The triple combination further enhanced apoptosis in 3/5 cell lines, suggesting these cell lines are likely dependent on both Bcl-xL and MCL1 for survival. Collectively, these findings demonstrate that combining BRAF-inhibitors with Bcl-xL and/or MCL1 inhibitors may represent a novel strategy for treating BRAF-mutant CRC. Citation Format: Laura J. Jenkins, Fiona Chionh, Ian Y. Luk, Erinna F. Lee, Amardeep S. Dhillon, Niall Tebbutt, Walter D. Fairlie, John M. Mariadason. BRAF inhibitors synergize with BH3 mimetics to induce apoptosis in BRAF mutant colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2492.

Anti-neoplastic potential of ethyl-p-methoxycinnamate of Kaempferia galanga on oral cancer cell lines

Abstract Kaempferia galanga is one of the herbal plant that have many medicinal properties. The plant extract has also been reported to possess in vitro anti-cancer activities on various cancer cell lines such as human colon cancer SW620, cervical cancer C33A, and breast cancer MCF-7 cell lines. Ethyl-p-methoxycinnamate (EPMC), a major constituent of volatile oil of Kaempferia galanga has been attributed to its medicinal benefits. We report here for the first time the anticancer potential of EPMC on oral cancer cells derived from human oral cavity, HSC-3 and Ca922 lines. MTT assay results showed that EPMC markedly induced cytotoxicity on HSC-3 (IC50 0.075 mg/mL) and Ca922 (IC50 0.085 mg/mL) cell lines. The apoptotic activity induced by EPMC was detected in both cell lines by flow cytometry and confirmed with Caspase 3/7 assay. Moreover, quantitative RT-PCR analysis revealed that EPMC-induced apoptosis in both lines elevated the mRNA levels of pro-apoptotic genes caspase-9 and PUMA. These results suggest that EPMC may be a potential candidate for oral cancer treatment.

Water-soluble Ru(II)-anethole compounds with promising cytotoxicity toward the human gastric cancer cell line AGS

AimsRuthenium-based compounds exhibit critical biochemical properties making them suitable for diverse pharmacological applications. The aim of this work was to study the anticancer effects of three ruthenium complexes on a human gastric cancer cell line. Main methodsWe synthetized three [Ru(η6-anethole)(en)X]PF6 complexes, where (en) is ethylenediamine and X is Cl (1), Br (2) or I (3), which were then evaluated by MTT assay, RT-qPCR and flow cytometry on the human gastric cancer cell line AGS. Key findingsCompound 3 exhibited the highest cytotoxicity (IC50 = 11.27 ± 1.08 μM) of the series, with an activity almost three-fold more potent than the commercial drug cisplatin, and also revealed a 4.5-fold less potent cytotoxicity in the human normal gastric cell line GES-1. The exchange of the halogen (Cl, Br or I) on the organometallic compound slightly alters its solubility in PBS and lipophilicity (expressed as Log P). Studies of gene expression revealed that compound 3 induces a significant overexpression of the pro-apoptotic genes Caspase-3, PUMA and DIABLO in the gastric cancer cell line AGS after 6 h. In contrast, only PUMA was significantly overexpressed in the normal gastric cell line GES-1. Compound 3 induced the activation of multiple caspases in AGS cells: a sign of apoptosis. Characterization via single-crystal X-ray diffraction for compound 3 confirmed the key structural features for this type of organometallic complexes. SignificanceOur data suggests that compound 3 may be an interesting anticancer molecule for the treatment of gastric cancer.

PO-094 Persistent Myc activity prevents resolution of liver injury

IntroductionFas/CD95 is a member of the death receptor superfamily that can induce apoptosis and it functions as the key component of the extrinsic death pathway. This study reports on a relevant function of Fas death receptor that can regulate transcription factors p53, MYC, NF-κ B and pro-apoptotic gene PUMA, that are important in intrinsic death pathway in hepatocellular carcinoma (HCC).Material and methodsFor the in vivo study, genes Fas, and FasL from 111 samples (39 HCC, 30 peri-HCC, 31 cirrhosis, and 11 normal) from patients undergoing liver resection without any prior treatments were analysed. For the in vitro study, hepatocytes cell line IHH and human HCC cell lines HepG2, HUH7, JHH6 were used. Apoptosis-induction was performed by using anti-Fas (DX2) at a concentration of 250 ng/ml and 500 ng/ml for 24 hours. Flow cytometry and quantitative real-time PCR were performed to analyse the data.Results and discussionThe expressions of Fas and FasL mRNA were significantly noticed in HCC as compared to normal tissues. FasL mRNA was high expressed in viral-related compared to metabolic-related HCC while Fas was high in HCC tissues without vascular invasion compared to tissues with vascular invasion. For in vitro study, after treatment of anti-CD95, TNF-α was significantly up regulated in HEPG2 compare to IHH cells. Transcription factors p53, MYC and NF-κ B were down regulated in HEPG2 cells while up regulated or no difference change in IHH cells. Pro-apoptotic gene Puma was only up regulated in IHH cell line. Telomerase was up regulated in IHH and significantly down regulated in HEPG2.ConclusionsWe observed a modulation of transcription factors p53, MYC, NF-κ B and pro-apoptotic genes Puma, TNF-α, and Telomerase after induction by CD95 antibody in acute phase treatment for 24 hours. The expression of PUMA and MYC were regulated by the transcription factors p53, thus suggesting the involvement of Fas in p53-dependent apoptosis pathway.

Abstract 3699: Histone deacetylase inhibitor ITF2357 induces apoptosis and increases doxorubicin cytotoxicity in preclinical models of human sarcoma

Abstract Sarcomas are rare malignancies with generally poor prognosis, for which current therapies have shown limited efficacy. In order to improve the prognosis of patients, there is an urgent medical need to identify new molecular targets that can be exploited for the development of innovative treatment approaches. Inhibition of histone deacetylases (HDACs) has emerged as a potential strategy to reverse aberrant epigenetic changes associated with cancer, and HDAC inhibitors (HDACi) have been reported as potent and specific anticancer molecules in preclinical and clinical studies. Here we investigated the molecular and functional effects of a new generation HDACi, ITF2357, in preclinical models of soft tissue and bone sarcomas. A wide panel of sarcomas cells, both established and patient derived cell lines, were used. For in vitro experiments Western Blot, RT-PCR, flow cytometry and conservative isobologram analysis have been performed. In vivo efficacy of ITF2357/doxorubucin combination was evaluated in nude mice bearing sarcoma xenografts. Using in vitro and in vivo sarcoma models, we demonstrate that ITF2357 decreases cell viability, activates apoptosis and increases the growth inhibitory efficacy of doxorubicin. The molecular mechanisms of action of ITF2357-mediated cell death implied the activation of mitochondrial apoptosis, as attested by the increase of pro-apoptotic BH3-only proteins, and both caspase and bcl-2 dependent cell death. We also found that ITF2357 transcriptionally upregulated the pro-apoptotic BH3-only genes Bax, Puma, Noxa and BIM in cells harbouring mutant or wild type p53, thus indicating a p53 independent mechanism. Both bcl-2 overexpression and caspases inhibition strongly reduced apoptosis, corroborating the evidence that activation of mitochondrial pathway occurs upstream and before caspases activation. We also found that ITF2357 induces a canonical autophagic process, and that inhibition of autophagy increased apoptosis induced by ITF2357, indicating that in our models autophagy shows a prosurvival effect. We also reported that ITF2357 induced FOXO1/3a up-regulation, FOXO proteins nuclear accumulation, and transcriptional activation of FOXO-target genes, such as BH3-only proteins Puma, Noxa and Bim. Knockdown experiments demonstrated that reduction of both FOXO1 and 3a did not inhibit the induction of Bim expression in response to ITF2357, and consequently did not protect sarcoma cells against ITF2357-induced apoptosis, independently from p53 status. Notably, ITF2357 synergized with doxorubicin to induce in vitro cell death and to reduce in vivo tumor growth. Therefore ITF2357 may represent an important therapeutic agent against human sarcoma regardless p53 status, and the pharmacological combination of ITF2357 with doxorubicin has the potential to enhance sensitization in different preclinical models of sarcoma. Citation Format: Maria Grazia Tupone, Marta Di Martile, Marianna Desideri, Simonetta Buglioni, Barbara Antoniani, Carlotta Mastroiorio, Rita Falcioni, Virginia Ferraresi, Nicola Baldini, Roberto Biagini, Michele Milella, Daniela Trisciuoglio, Donatella Del Bufalo. Histone deacetylase inhibitor ITF2357 induces apoptosis and increases doxorubicin cytotoxicity in preclinical models of human sarcoma [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 3699.

Abstract 196: The effect of modulated electro-hyperthermia on B16-F10 melanoma tumor growth

Abstract The objective of the present study was to examine the effect of modulated electro-hyperthermia (mEHT) on B16-F10 mouse melanoma cell line in vitro and in vivo. mEHT induces tumor-selective heat shock at ~42°C and is used as adjuvant to conventional cancer therapy. For in vitro comparison of the effect of mEHT to conventional hyperthermia (convHT), cells grown on coverslips were treated using mEHT or convHT in water-bath at 42°C for 1x60 min. Changes in gene expression, viability and invasiveness were measured. For in vivo studies, B16-F10 cells mixed with Matrigel were injected subcutaneously into C57Bl/6 mice as the tumors were exposed to 42°C for 30 minutes with mEHT every second day for three times. Tumors were removed 48 hours post treatment, weighed and digested to single-cell suspension. The supernatant of tumor cells and the plasma of tumor-bearing mice were used to screen a membrane-based antibody array representing a panel of 111 cytokines. The tumor cells were stained for flow cytometry to determine the changes induced on melanoma cells and tumor-infiltrating leukocytes. As a marker of in vitro treatment we measured HSPA2 expression with qPCR. Similar expression patterns were induced both by mEHT and convHT. Three hours after treatment the peak levels of HSPA2 were 3 times higher as compared to the untreated control in a time-course experiment. Next, we showed that in parallel to the upregulation of proapoptotic genes Puma, Bak-1 and Bax, downregulation of prosurvival genes XIAP, Bcl-2, Bcl-XL was induced both by convHT and mEHT with similar kinetics and magnitude. Furthermore, cell viability decreased and the invasivness was impaired as measured by scratch assay by both treatment types. The in vivo mEHT of primary melanoma tumors resulted in considerable tumor size reduction. Flow cytometry analysis revealed the increase of MHC class I on the surface of melanoma cells and concomitant activation of CD8+ cytotoxic T lymphocytes in response to treatment. Increased number of CD4+ T cells and Gr1+ granulocytes was detected, although changes in the various subpopulations need further analysis. F4/80+CD11b+ macrophages and mature DC-s were slightly elevated in the treated tumors. The cytokine array analysis shows that several cytokines responsible for leukocyte recruitment were enriched in the treated tumor (SDF-1, osteopontin, IL-1). Complement activation was induced by mEHT both in the tumor microenvironment and the plasma, indicating an acute inflammatory response to treatment. Furthermore, along the short pentraxins, CRP and SAP, the long pentraxin PTX3 was elevated in the tumor and plasma of the treated mice, suggesting that mEHT induced the tumor clearance. Taken together, our results demonstrate that in vitro mEHT and convHT produce comparable effects, whereas the in vivo mEHT treatment was effective in reducing the tumor size by enhancing immune recognition of B16-F10. This study was supported by NVKP 16-1- 2016-0042 grant. Citation Format: Balázs Besztercei, Enikő Major, Anett Benedek, Zoltán Benyó, Andrea Balogh. The effect of modulated electro-hyperthermia on B16-F10 melanoma tumor growth [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 196.

Involvement of proapoptotic genes in autophagic cell death induced by irradiation

Irradiation is widely used in anticancer therapy; however, the efficiency is limited. Most cancer cells have mutations in apoptosis that they can easily escape the apoptosis induced by irradiation. Autophagy has been known as type II programmed cell death that can be activated by irradiation, especially when apoptosis is blocked, but the underlying molecular mechanism is largely unknown. We demonstrated that proapoptotic genes PUMA and Bid are involved in the regulation of autophagic cell death. When wild-type (WT), Bax−/− and PUMA−/− HCT116 cells were exposed to irradiation, we found that, compared with WT, Bax−/− cells showed significantly decreased cell death because of Bax deficiency; however, surprisingly PUMA−/− cells showed significant increase in cell death although the proapoptotic gene PUMA was knocked out. By analyzing apoptosis via Annexin V-fluorescein isothiocyanate (FITC) assay with flow cytometry, both Bax−/− and PUMA−/− cells showed less apoptosis than WT, suggesting the existence of another type of cell death in PUMA−/− cells. Autophagy was then examined in three cell lines by counting the percentage of cells with punctate GFP-LC3. Although all three cell lines showed significantly increased autophagy activity after irradiation, that of PUMA−/− cells was much higher than the other two cell lines, which suggests that PUMA−/− cells may die through autophagy. This was then confirmed by the decreased cell death in PUMA−/− cells when autophagy was blocked by 3-MA. In addition, we also tested the responses of WT and Bid−/− MEFs to irradiation. Bid−/− MEFs but not WT died through autophagy after irradiation. These results imply the involvement of apoptosis-associated genes such as PUMA and Bid in autophagic cell death, which contributes to identifying the molecular mechanism by which autophagy drives cells to death.

The Functional Roles of the MDM2 Splice Variants P2-MDM2-10 and MDM2-∆5 in Breast Cancer Cells

BACKGROUND: MDM2 is a negative regulator of p53 and is upregulated in numerous human cancers. While different MDM2 splice variants have been observed in both normal tissues and malignant cells, their functions are poorly understood. METHODS: We evaluated the effect of MDM2 splice variants by overexpression in MCF-7 cells and analyses of expression of downstream genes (qPCR and Western blot), subcellular localization (immunofluorescence), cell cycle assays (Nucleocounter3000), apoptosis analysis (Annexin V detection), and induction of senescence (β-galactosidase analysis). RESULTS: In a screen for MDM2 splice variants in MCF-7 breast cancer cells, extended with data from healthy leukocytes, we found P2-MDM2-10 and MDM2-Δ5 to be the splice variants expressed at highest levels. Contrasting MDM2 full-length protein, we found normal tissue expression levels of P2-MDM2-10 and MDM2-Δ5 to be highest in individuals harboring the promoter SNP309TT genotype. While we detected no protein product coded for by MDM2-Δ5, the P2-MDM2-10 variant generated a protein markedly more stable than MDM2-FL. Both splice variants were significantly upregulated in stressed cells (P=4.3 × 10−4 and P=7.1 × 10−4, respectively). Notably, chemotherapy treatment and overexpression of P2-MDM2-10 or MDM2-Δ5 both lead to increased mRNA levels of the endogenous MDM2-FL (P=.039 and P=.070, respectively) but also the proapoptotic gene PUMA (P=.010 and P=.033, respectively), accompanied by induction of apoptosis and repression of senescence. CONCLUSION: We found P2-MDM2-10 and MDM2-Δ5 to have distinct biological functions in breast cancer cells. GENERAL SIGNIFICANCE: Alternative splicing may influence the oncogenic effects of the MDM2 gene.

The Effects of Silica Nanoparticles on Apoptosis and Autophagy of Glioblastoma Cell Lines.

Silica nanoparticles (SiNPs) are one of the most commonly used nanomaterials in various medical applications. However, possible mechanisms of the toxicity caused by SiNPs remain unclear. The study presented here provides novel information on molecular and cellular effects of SiNPs in glioblastoma LBC3 and LN-18 cells. It has been demonstrated that SiNPs of 7 nm, 5–15 nm and 10–20 nm induce time- and dose-dependent cytotoxicity in LBC3 and LN-18 cell lines. In contrast to glioblastoma cells, we observed only weak reduction in viability of normal skin fibroblasts treated with SiNPs. Furthermore, in LBC3 cells treated with 5–15 nm SiNPs we noticed induction of apoptosis and necrosis, while in LN-18 cells only necrosis. The 5–15 nm SiNPs were also found to cause oxidative stress, a loss in mitochondrial membrane potential, and changes in the ultrastructure of the mitochondria in LBC3 cells. Quantitative real-time PCR results showed that in LBC3 cells the mRNA levels of pro-apoptotic genes Bim, Bax, Puma, and Noxa were significantly upregulated. An increase in activity of caspase-9 in these cells was also observed. Moreover, the activation of SiNP-induced autophagy was demonstrated in LBC3 cells as shown by an increase in LC3-II/LC3-I ratio, the upregulation of Atg5 gene and an increase in AVOs-positive cells. In conclusion, this research provides novel information concerning molecular mechanisms of apoptosis and autophagy in LBC3 cells.

PUMA gene delivery to synoviocytes reduces inflammation and degeneration of arthritic joints

In rheumatoid arthritis (RA), the proliferation of fibroblast-like synoviocytes (FLS) is the cause of chronic inflammation in joints and of joint damage. Delivery of the pro-apoptotic gene PUMA to FLS via human adenovirus type 5 (HAdV5) vectors has been tested as a therapeutic approach, but efficiency is hampered by low transduction, as FLS do not express HAdV5 receptors on the cell surface. Here we show that efficient transduction of PUMA in FLS can be achieved by conjugating HAdV5 to a baculovirus, which binds to the cell surface via the envelope glycoprotein Gp64. Intra-articular injection in an adjuvant-induced rat model of RA induces apoptosis of FLS, leading to significant decrease in joint inflammation, joint damage, and bone loss with improvement in joint function and mobility. Our results demonstrate the therapeutic potential of PUMA gene therapy as a local treatment in various forms of arthritis in which abnormal FLS proliferation is implicated.

Testosterone modulates FoxO3a and p53-related genes to protect C2C12 skeletal muscle cells against apoptosis

The loss of muscle mass and strength with aging, sarcopenia, is a prevalent condition among the elderly, associated with skeletal muscle dysfunction and enhanced muscle cell apoptosis. We have previously demonstrated that testosterone protects against H2O2-induced apoptosis in C2C12 muscle cells, at different levels: morphological, biochemical and molecular. Since we have observed that testosterone reduces p-p53 and maintains the inactive state of FoxO3a transcription factor, induced by H2O2, we analyzed if the hormone was exerting its antiapoptotic effect at transcriptional level, by modulating pro and antiapoptotic genes associated to them. We detected the upregulation of the proapoptotic genes Puma, PERP and Bim, and MDM2 in response to H2O2 at different periods of the apoptotic process, and the downregulation of the antiapoptotic gene Bcl-2, whereas testosterone was able to modulate and counteract H2O2 effects. Furthermore, ERK and JNK kinases have been demonstrated to be linked to FoxO3a phosphorylation and thus its subcellular distribution. This work show some transcription level components, upstream of the classical apoptotic pathway, that are activated during oxidative stress and that are points where testosterone exerts its protective action against apoptosis, exposing some of the puzzle pieces of the intricate network that aged skeletal muscle apoptosis represents.

Reevaluation of ATR signaling in primary resting chronic lymphocytic leukemia cells: evidence for pro-survival or pro-apoptotic function.

ATM, primarily activated by DNA double-strand breaks, and ATR, activated by single-stranded DNA, are master regulators of the cellular response to DNA damage. In primary chronic lymphocytic leukemia (CLL) cells, ATR signaling is considered to be switched off due to ATR downregulation. Here, we hypothesized that ATR, though expressed at low protein level, could play a role in primary resting CLL cells after genotoxic stress. By investigating the response of CLL cells to UV-C irradiation, a prototypical activator of ATR, we could detect phosphorylation of ATR at Thr-1989, a marker for ATR activation, and also observed that selective ATR inhibitors markedly decreased UV-C-induced phosphorylation of ATR targets, including H2AX and p53. Similar results were obtained with the purine analogs fludarabine and cladribine that were also shown to activate ATR and induce ATR-dependent phosphorylation of H2AX and p53. In addition, ATR inhibition was found to sensitize primary CLL cells to UV-C by decreasing DNA repair synthesis. Conversely, ATR inhibition rescued CLL cells against purine analogs by reducing expression of the pro-apoptotic genes PUMA and BAX. Collectively, our study indicates that ATR signaling can be activated in resting CLL cells and play a pro-survival or pro-apoptotic role, depending on the genotoxic context.