Strong Cytotoxic Effect Research Articles

Combined Effect of Vaccinium nilgiriensis Bark Extract and 680nm Laser Irradiation in Inducing Breast Cancer Cell Death.

Cancer refers to a collection of diseases where cells begin to multiply uncontrollably. Breast cancer is the most predominant malignancy in women. Herbal medicine is one of the important health care systems in most developing countries. Many studies have shown that naturally occurring compounds may support the prevention and treatment of various diseases, including cancer. Some of the plant extracts and isolated compounds show photosensitizing activities and reduce cell proliferation whereas some have revealed photoprotective effects. The biological properties and medicinal uses of extracts and bioactive compounds from V. nilgiriensis have not been investigated. This study aims to evaluate the cytotoxic effects of V. nilgiriensis in combination with 680nm laser irradiation on MCF-7 breast cancer cells. The inverted microscopy, ATP and LDH assay were used to analyze the cellular morphology, proliferation, cytotoxicity respectively after the treatment with V. nilgiriensis bark extract. The diode laser of wavelength 680nm and 15 J/cm2 fluency has been used for laser irradiation. The activity of apoptotic proteins was studied using ELISA and nuclear damage by Hoechst staining. The exposure of V. nilgiriensis extracts with laser irradiation at 680nm increases the cytotoxicity and decreases the proliferation of MCF-7 cells. The results of the Hoechst stain indicated nuclear damage. Our study proved that V. nilgiriensis holds a strong cytotoxic effect on breast cancer cells alone and in combination with laser irradiation by upregulating the expression of apoptotic proteins such as caspase 3, p53 and Bax. The results from this study showed that the bark ethyl acetate of V. nilgiriensis and in combination with laser is effective in preventing breast cancer cell proliferation in vitro. Further work is warranted to isolate the bioactive compounds from V. nilgiriensis bark extract and study the effect of compounds in the cell death induction. Due to the cytotoxic properties, V. nilgiriensis can be considered as a potent therapeutic agent for the treatment of cancer.

Identification of Novel Small-Molecule Kinase Modulators for the Treatment of Neuroblastoma.

Neuroblastoma represents 8–10% of all childhood cancer cases and is responsible for 15% of all cancer-related deaths in infants. Even though patients with low- and intermediate-risk disease have a good prognosis, the 5-year survival rate of the vast majority of patients with high-risk neuroblastoma is 50%. Despite extensive research efforts to find a cure for neuroblastoma, current treatment options are still limited. The aim of our study was to identify novel therapeutic compounds using high-throughput drug screening of a small molecule kinase inhibitor library containing 960 compounds. This screening resulted in the identification of two compounds, ST013381 and ST022328, that showed pronounced cytotoxic effects in six human neuroblastoma cell lines in vitro while having reduced effects in the BJ-5ta control cell line. These effects were observed in both MYCN-amplified and -non-amplified cells, indicating that these compounds can affect a wide range of neuroblastomas. Our experiments also revealed that several signaling pathways underlie the selective elimination of neuroblastoma cells by the ST013381 and ST022328 compounds. In summary, we have identified two novel compounds with a strong cytotoxic effect in vitro as promising agents for the treatment of neuroblastoma.Electronic Supplementary MaterialThe online version of this article (10.1007/s40487-020-00113-5) contains supplementary material, which is available to authorized users.

Cytotoxic Effects of Plant Sap-Derived Extracellular Vesicles on Various Tumor Cell Types.

Edible plants have been widely used in traditional therapeutics because of the biological activities of their natural ingredients, including anticancer, antioxidant, and anti-inflammatory properties. Plant sap contains such medicinal substances and their secondary metabolites provide unique chemical structures that contribute to their therapeutic efficacy. Plant extracts are known to contain a variety of extracellular vesicles (EVs) but the effects of such EVs on various cancers have not been investigated. Here, we extracted EVs from four plants—Dendropanax morbifera, Pinus densiflora, Thuja occidentalis, and Chamaecyparis obtusa—that are known to have cytotoxic effects. We evaluated the cytotoxic effects of these EVs by assessing their ability to selectively reduce the viability of various tumor cell types compared with normal cells and low metastatic cells. EVs from D. morbifera and P. densiflora sap showed strong cytotoxic effects on tumor cells, whereas those from T. occidentalis and C. obtusa had no significant effect on any tumor cell types. We also identified synergistic effect of EVs from D. morbifera and P. densiflora saps on breast and skin tumor cells and established optimized treatment concentrations. Our findings suggest these EVs from plant sap as new candidates for cancer treatment.

Modulating the dose-rate differently affects the responsiveness of human epithelial prostate- and mesenchymal rhabdomyosarcoma-cancer cell line to radiation

Purpose: Radiation therapy (RT), by using ionizing radiation (IR), destroys cancer cells inducing DNA damage. Despite several studies are continuously performed to identify the best curative dose of IR, the role of dose-rate, IR delivered per unit of time, on tumor control is still largely unknown.Materials and methods: Rhabdomyosarcoma (RMS) and prostate cancer (PCa) cell lines were irradiated with 2 or 10 Gy delivered at dose-rates of 1.5, 2.5, 5.5 and 10.1 Gy/min. Cell-survival rate and cell cycle distribution were evaluated by clonogenic assays and flow cytometry, respectively. The production of reactive oxygen species (ROS) was detected by cytometry. Quantitative polymerase chain reaction assessed the expression of anti-oxidant-related factors including NRF2, SODs, CAT and GPx4 and miRNAs (miR-22, -126, -210, -375, -146a, -34a). Annexin V and caspase-8, -9 and -3 activity were assessed to characterize cell death. Senescence was determined by assessing β-galactosidase (SA-β-gal) activity. Immunoblotting was performed to assess the expression/activation of: i) phosphorylated H2AX (γ-H2AX), markers of DNA double strand breaks (DSBs); ii) p19Kip1/Cip1, p21Waf1/Cip1 and p27Kip1/Cip1, senescence-related-markers; iii) p62, LC3-I and LC3-II, regulators of autophagy; iv) ATM, RAD51, DNA-PKcs, Ku70 and Ku80, mediators of DSBs repair.Results: Low dose-rate (LDR) more efficiently induced apoptosis and senescence in RMS while high dose-rate (HDR) necrosis in PCa. This paralleled with a lower ability of LDR-RMS and HDR-PCa irradiated cells to activate DSBs repair. Modulating the dose rate did not differently affect the anti-oxidant ability of cancer cells.Conclusion: The present results indicate that a stronger cytotoxic effect was induced by modulating the dose-rate in a cancer cell-dependent manner, this suggesting that choose the dose-rate based on the individual patient’s tumor characteristics could be strategic for effective RT exposures.

MAY, a novel tubulin inhibitor, induces cell apoptosis in A549 and A549/Taxol cells and inhibits epithelial-mesenchymal transition in A549/Taxol cells

Non-small-cell lung cancer (NSCLC) is one of the common malignant tumors, and multidrug resistance (MDR) and tumor metastasis limit the anticancer effect of NSCLC. Therefore, it is necessary to develop new anticancer drug that can inhibit MDR and metastasis of NSCLC. In the present study, we found that 5-(2-chlorophenyl)-4-(4-(3,5-dimethoxyphenyl)piperazine-1-carbonyl)-2H-1,2,3- triazole (MAY) displayed strong cytotoxic effect on A549 and taxol-resistant A549 cells (A549/Taxol cells). We further discovered that MAY led to G2/M phase arrest by inhibiting microtubule polymerization in both cells. Then MAY caused apoptosis by the mitochondrial pathway in A549 cells and through the extrinsic pathway in A549/Taxol cells. Interestingly, MAY was not a substrate for P-glycoprotein (P-gp), which was highly expressed in A549/Taxol cells, and MAY inhibited the expression and efflux function of P-gp. Furthermore, MAY inhibited epithelial-mesenchymal transition (EMT) by targeting Twist1 in A549/Taxol cells. In summary, our results suggest that MAY induces apoptosis in A549 and A549/Taxol cells and inhibits EMT in A549/Taxol cells. These findings suggest that MAY could provide a promising method for the treatment of NSCLC, especially for the treatment of resistant NSCLC.

Novel ruthenium and palladium complexes as potential anticancer molecules on SCLC and NSCLC cell lines

Lung cancer is one of the major causes of cancer-related deaths in the world. Non-small-cell lung cancer (NSCLC) is the most common type of lung cancer, and small-cell lung cancer (SCLC) is the most aggressive subtype of lung cancer. Proper therapies for SCLC have not yet been developed. However, new molecules have been designed and big innovation in treating SCLC has been achieved. Platinum-based antitumor drugs like cisplatin and carboplatin have several disadvantages including side effects, cisplatin-resistant tumors and limited solubility in aqueous media. Thus, two novel chiral aminoalcohol-based bis(phosphinite) ligands containing (η6-p-cymene)-Ru(II)-phosphinite and bis(phosphinite)–Pd(II) complexes were synthesized and evaluated for anticancer activity. In this study, the results showed that complex 1 has the strongest cytotoxic effects on SCLC and NSCLC cell lines. On the other hand, cisplatin, ruthenium and palladium complexes are capable to induce apoptosis. Especially, complexes 1 and 2 can induce apoptosis for both SCLC and NSCLC. When compared to the qRT-PCR and TUNEL results, we obtained a significant correlation between apoptotic index and p21, Bax gene expressions. This work revealed the potential of the synthesized complexes as anticancer agents with cytotoxic and pro-apoptotic activity as leading compounds for further anticancer researches.

Evaluating the antitumor activity of sphingosine-1-phosphate against human triple-negative breast cancer cells with basal-like morphology

Sphingosine-1-phosphate (S1P) is an important sphingolipid metabolite that regulates a wide range of physiological and pathophysiological processes. Our previous studies show that S1P selectively induces cell apoptosis in human breast cancer luminal A subtype cell line MCF7. In addition, S1P exhibits synergistic effects with chemotherapy drugs against both MCF7 and luminal B subtype cell line MDA-MB-361 at concentration in the high nM to low μM range. In the current study, we evaluated the effect of S1P on proliferation, apoptosis and cytotoxicity towards a panel of nine triple-negative breast cancer with basal-like morphology (TNBC-BL) cell lines (HCC1599, HCC1937, HCC1143, MDA-MB-468, HCC38, HCC70, HCC1806, HCC1187 and DU4475) in the same concentration range. S1P exhibited mild to moderate effects (<20% increase comparted to control) towards the TNBC-BL cell lines except HCC38, HCC70 and HCC1806. Furthermore, it increased cell apoptosis by ~15-20% in all the cell lines compared to the control, and elicited moderate to strong cytotoxic effect towards all cell lines except MDA-MB-468 and HCC1806. However, no synergistic/additive effect was observed between S1P and chemotherapy drug docetaxel for any TNBC-BL cell line.

Seleninic Acid Potassium Salts as Water-Soluble Biocatalysts with Enhanced Bioavailability.

Organoselenium compounds are well-known glutathione peroxidase (GPx) mimetics that possess antioxidants/prooxidant properties and are able to modulate the concentration of reactive oxygen species (ROS), preventing oxidative stress in normal cells or inducing ROS formation in cancer cells leading to apoptosis. The purpose of this study was the synthesis of potent GPx mimics with antioxidant and anticancer activity along with improved bioavailability, as a result of good solubility in protic solvents. As a result of our research, glutathione peroxidase (GPx) mimetics in the form of water-soluble benzeneseleninic acid salts were obtained. The procedure was based on the synthesis of 2-(N-alkylcarboxyamido)benzeneselenenic acids, through the oxidation of benzisoselenazol-3(2H)-ones or analogous arenediselenides with an amido group, which were further converted to corresponding potassium salts by the treatment with potassium tert-butanolate. All derivatives were tested as potential antioxidants and anticancer agents. The areneseleninic acid salts were significantly better peroxide scavengers than analogous acids and the well-known organoselenium antioxidant ebselen. The highest activity was observed for the 2-(N-ethylcarboxyamido)benzeneselenenic acid potassium salt. The strongest cytotoxic effect against breast cancer (MCF-7) and human promyelocytic leukemia (HL-60) cell lines was found for 2-(N-cyclohexylcarboxyamido)benzeneselenenic acid potassium salt and the 2-(N-ethylcarboxyamido)benzeneselenenic acid, respectively. The structure–activity correlations, including the differences in reactivity of benzeneseleninic acids and corresponding salts were evaluated.

Cytotoxicity of Alstonia scholaris (R.Br) bark on MCF-7 and Vero cell lines

Alstonia scholaris R.Br (pulai) is one of traditional forest plant to treat many kinds of disease such as arthritis, diabetes, hypertension, and hyperlipidaemia. On the other hand research about this species as anti cancer is very limited. This study aimed to determine in vitro cytotoxic activity of three fraction of A. Scholaris bark against breast cancer (MCF-7) and normal (vero) cells. Three fractions of A. Scholaris bark (n-hexane, chloroform, and ethanol fractions) derived from 70% ethanol crude extracted pulai bark. MTT (3- 4,5-dimethylthiazol-2- -2,5-diphenyl tetrazolium bromide) assay applied tot MCF-7 and vero cells. The result showed that n-hexane fraction had the strongest cytotoxic effect on MCF-7 cells, followed by chloroform and ethanol fraction with IC50 values 109.01; 163.33; and 264.19 µg/mL respectively, but ethanol fraction is the least toxic on vero cells growth compared to n-hexane and chloroform fraction with IC50 value 579.93; 459.47; and 396.24 µg/mL respectively. It’s suggested that ethanol fraction is the best recommended fraction as anticancer agent because it was the least toxicity to the growth of normal cells, but still toxic to cancer cell. It was concluded that ethanol fraction was the best fraction to breast cancer cells.

Basic Investigations of Singlet Oxygen Detection Systems with ESR for the Measurement of Singlet Oxygen Quenching Activities.

Singlet oxygen (1O2) is highly oxidative and exerts strong cytotoxic effects. We tried to establish the best combination of a singlet oxygen generation system and a detection method with ESR, for measurement of the quenching activities of various substances. The photosensitizing reaction of rose bengal or thermal decomposition of 4-methyl-1,4-etheno-2,3-benzodioxin-1(4H)-propanoic acid (endoperoxide, EP) was used for the generation of 1O2, and a sterically hindered secondary amine, 2,2,6,6-tetramethyl-4-piperidone (TEMPD) or 2,2,6,6-tetramethyl-4-piperidinol (TEMP-OH), was used as the 1O2 detection probe. These secondary amines were oxidized by 1O2 to form stable nitroxide radicals, which were detectable by ESR. TEMPD was found to be readily oxidized by air, causing large background signals in comparison with TEMP-OH. The ESR signal obtained by the irradiation of rose bengal with visible light in the presence of TEMP-OH consisted of two kinds of nitroxide radical overlapping. In contrast, only a single nitroxide signal was observed when TEMP-OH was reacted with 1O2 generated from EP. Therefore, the best combination should be EP as the 1O2 generator and TEMP-OH as the detection probe. When using this combination, we found that the concentrations of some organic solvents such as dimethyl sulfoxide and acetonitrile should be kept constant for reliable quantification, because the concentrations of organic solvents affect the ESR signal intensity.

Isopanduratin A Isolated from Boesenbergia pandurata Reduces HepG2 Hepatocellular Carcinoma Cell Proliferation in Both Monolayer and Three-Dimensional Cultures.

Medicinal plants have been used for disease treatment throughout history, especially in parts of Asia. Vietnam is a tropical country which possesses forests with a wide diversity of plants, which have a long history of being used as alternative remedies for treatment of various diseases. In this study, we aimed to test the bioactivity of Boesenbergia pandurata (B. pandurata) plant extract-derived compounds, including Pinostropin, Pinocembrin, Alpinetin, and Isopanduratin A, on HepG2 hepatocellular carcinoma cells. In this study, several compounds from Boesenbergia pandurata plant extract, including Pinostropin, Pinocembrin, Alpinetin, and Isopanduratin A, were used to evaluate their antitumor effects on HepG2 hepatocellular carcinoma cells in both monolayer culture condition (2D culture) and three-dimensional culture condition (3D culture); HepG2 cell proliferation was assessed by Alamar Blue assay. The compound which had the strongest cytotoxicity was selected for further analysis by Propidium Iodide (PI) staining assay and caspase 3/7 expression assay. The cytotoxicity assay showed that Isopanduratin A had the strongest cytotoxic effect on HepG2 cells grown in both 2D culture (IC50 at 357±10nM) and 3D culture conditions (IC50 at 744±18nM). The PI staining of HepG2 spheroids demonstrated that Isopanduratin A was potently cytotoxic to HepG2 cells growing in spheroids (3D) and destroyed the initial shape of the spheroids. Isopanduratin A was shown to induce caspase 3/7 after 30min of incubation at the concentration of 500nM. This study demonstrated that the compound Isopanduratin A isolated from B. pandurata extract has a strong inhibitory effect on cell growth in 2D and 3D culture conditions. Isopanduratin A also has the ability to induce apoptosis in HepG2 cells via activation of caspase 3/7. Further investigations on the mechanisms of Isopanduratin A-induced effects on HepG2 cells, and other cancer cells, are necessary.

Lactoferrin-tethered betulinic acid nanoparticles promote rapid delivery and cell death in triple negative breast and laryngeal cancer cells

Cancer management presents multifarious problems. Triple negative breast cancer (TNBC) is associated with inaccurate prognosis and limited chemotherapeutic options. Betulinic acid (BA) prevents angiogenesis and causes apoptosis of TNBC cells. NIH recommends BA for rapid access in cancer chemotherapy because of its cell-specific toxicity. BA however faces major challenges in therapeutic practices due to its limited solubility and cellular entree. We report lactoferrin (Lf) attached BA nanoparticles (Lf-BAnp) for rapid delivery in triple negative breast (MDA-MB-231) and laryngeal (HEp-2) cancer cell types. Lf association was confirmed by SDS-PAGE and FT-IR analysis. Average hydrodynamic size of Lf-BAnp was 147.7 ± 6.20 nm with ζ potential of −28.51 ± 3.52 mV. BA entrapment efficiency was 75.38 ± 2.70% and the release mechanism followed non-fickian pattern. Impact of Lf-BAnp on cell cycle and cytotoxicity of triple negative breast cancer and its metastatic site laryngeal cancer cell lines were analyzed. Lf-BAnp demonstrated strong anti-proliferative and cytotoxic effects, along with increased sub-G1 population and reduced number of cells in G1 and G2/M phases of the cell cycle, confirming reduced cell proliferation and significant cell death. Speedy intracellular entry of Lf-BAnp occurred within 30 min. Lf-BAnp design was explored for the first time as safer chemotherapeutic arsenals against complex TNBC conditions.

Chemical compositions and antiproliferative effect of essential oil of asafoetida on MCF7 human breast cancer cell line and female wistar rats

Background: Breast cancer is a leading cause of cancer-associated mortality in women, and the incidence is on the rise worldwide. Asafoetida has shown a good anti-cancer activity against breast cancer in both in vivo and in vitro studies. Materials and Methods: For the evaluation of the cytotoxic effect of essential oil of asafoetida (EOA), MCF7 cells, a highly invasive variant of human breast cancer cell line, were exposed to different concentrations of EOA (2, 4, 6, 8, and 10 μl/ml) over different periods (24, 48, and 72 h), followed by cell viability analysis using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. For determining thein vivo toxicity of EOA, the oil was administered orally at doses of 0.1, 1, 10, and 100 μl/kg for 28 days in female Wistar rats. After completion of the experiment, hematological and serum biochemical parameters were evaluated and compared to the untreated group. Results: MTT assay results showed that EOA significantly decreased the viability of MCF7 cells in a time- and concentration-dependent manner, demonstrating a strong cytotoxic effect of EOA on breast cancer cells. In chronic toxicity study, the female Wistar rats showed no change in hematological and biochemical parameters at any of the administered dose. Conclusions: The cytotoxic effect of EOA on breast cancer cells, without general toxicity, makes it a promising compound as adjuvant therapy for breast cancer.

Synthesis, crystal structures, spectroscopic, and cytotoxicity study of Cu(II), Co(II), Ni(II) and Pd(II) complexes with 2-anilinomethylidene-5,5-dimethylcyclohexane-1,3-dione

The coordination compounds of Cu(II), Co(II), Ni(II) and Pd(II) with 2-anilinomethylidene-5,5-dimethylcyclohexane-1,3-dione (HL) of the composition [ML2(C2H5OH)2]·nH2O (M = Co (n = 1), Ni (n = 0)) and [ML2] (M = Cu, Pd) have been prepared and investigated by elemental analysis, IR-spectroscopy, NMR spectroscopy, powder X-ray diffraction analysis. Single-crystal X-ray diffraction analysis shows that HL serves as a chelate bidentate ligand coordinated to the metal ion through the N and O atoms forming a six-membered chelate ring. The coordination polyhedron of the metal atoms is a square bipyramid [ML2(C2H5OH)2] or square planar [ML2]. The Cu(II) and Pd(II) complexes stability in solution has been shown by NMR spectroscopy. The results of the cell viability study on Hep2 (human larynx carcinoma) cells demonstrate that the complexes don’t exhibit strong cytotoxic effect against Hep-2 cell line compared to cisplatin (IC50 = 9,2 ± 0,5 μM). Palladium(II) complex is the most cytotoxic among all complexes and shows IC50 value of 68,7 ± 5,3 μM.

Chemical constituents from Schisandra sphenanthera and their cytotoxic activity

Extensive phytochemical investigation of Schisandra sphenanthera leaves resulted in the isolation of six highly oxygenated nortriterpenoids (1–6) and five lignans (7–11) including a new pre-schisanartane-type, schisandrathera A (1), a new dibenzocyclooctadiene glycoside, schisandrathera B (7) and two new lignans, schisandrathera C (8) and schisandrathera D (9). Their chemical structures including absolute configurations were determined extensively by means of HR-ESI-MS, NMR, and ECD spectra. In addition, all isolated compounds were tested for cytotoxic activity against PC3 (prostate cancer) and MCF7 (breast cancer) cell lines. Among these compounds, schirubrisin B (3) showed strong cytotoxic effect on both PC3 and MCF7 cell lines with IC50 values of 3.21 ± 0.68, 13.30 ± 0.68 μM, respectively, whereas ten remaining compounds were found to be less effective in the investigated models.

Study of the possible synergistic protective effects of Melatonin and Pregabalin in Vincristine induced peripheral neuropathy Wistar Albino rats

AimsInvestigate Melatonin administration along with Vincristine (VCR) owing to two reasons: First, Melatonin’s sciatic nerve protective effect against Chemotherapy Induced Peripheral Neuropathy (CIPN). Second, Vincristine’s anticancer work potentiation that may lead to a decrease in its dose and its side effects accordingly. Main methodsIn vivo and in vitro experiments were conducted in the study. The in vivo experiment included developing a CIPN Wistar Albino rat model. They were injected with VCR sulfate to induce CIPN, while Melatonin and Pregabalin (neuroleptic) were co-administered with the aim of investigating the degeneration/protection of the sciatic nerve. Tail Immersion Test and histopathological analyses were done to validate the experimental model (induction of rat peripheral neurodegeneration) and examine the possible Melatonin and Pregabalin protective/analgesic effects respectively. Liver and kidney function tests, catalase activity and malondialdehyde (MDA) level were measured. Concerning the in vitro experiment, cell viability assays in addition to ELISA (for caspases 3&9) were conducted using HCT-116 colon cancer cell line treated with VCR, Melatonin and their combination. Key findingsVCR caused an elevated level of oxidation (MDA level increment) in the sciatic nerve, while Melatonin could prove its antioxidant effect through increasing catalase activity (antioxidant enzyme) with no significant toxic effect on body's functions. HCT-116 in vitro test proved the strong cytotoxic effect of VCR in addition to the synergistic effect of combining Melatonin to VCR on the cellular apoptotic level (caspases 3&9 increased activity levels) and inhibitory concentration 50%. SignificanceMelatonin possesses an antioxidant property that can protect the sciatic nerve from CIPN-induced degeneration and potentiate VCR's anticancer effect at the same time.

Analog Curcumin, Pentagamavunon-0 (PGV-0), Induces Senescence and Increases Cytotoxic Effect of Doxorubicin on HCC 1954 Cells

Senescence defined as an irreversible cell cycle arrest. Senescence can inhibit cancer growth and suppress the progression of cancer. Some anticancer compounds are known to have the potential to induce senescence. Senescence defence against tumor development by preventing proliferation of cells with DNA damage. The study aimed to determine the cytotoxic effects and senescence induction of Pentagamavunon-0 (PGV-0) on Human Epidermal Growth Factor Receptor 2-positive (HER2-positive) breast cancer cells, HCC 1954. Cytotoxic tests carried out with 3- (4.5-dimethylthiazzol-2yl) -2.5-tidiphenyltetrazolium (MTT) assay showed that PGV-0 exhibited a strong cytotoxic effect with a the half maximal inhibitory concentration (IC50) value of 39 μM. Treatment with IC50 in sub-doses combined with doxorubicin showed cytotoxic enhancement effects. The senescence assay using SA-β-Galactosidase showed that the PGV-0 in a single treatment was able to induce senescence with a percentage of cell senescent of 15%. The combination treatment of PGV-0 at the half dose of IC50 with doxorubicin 100 nM was able to induce senescence with the percentage of senescent cells of 25%. Moreover, PGV-0 also increased intracellular reactive oxygen species (ROS). The results of this study indicate that PGV-0 exhibits cytotoxic effect, increases cytotoxic effect of doxorubicin and induces senescence that may correlate to the increasing of intracellular ROS in 1954 HCC cells.Keywords: Pentagamavunon (PGV-0), HCC 1954, Cytotoxic, Senescence

Abstract A018: Synergy between IAP inhibitors and a cytotoxic antibody-based chimeric protein in pancreatic cancer cells

Abstract Background: immunotoxins are cytotoxic antibody-toxin gene fusion proteins engineered to target cancer cells via antibody binding to surface antigens. Immunotoxins derived from Pseudomonas exotoxin inhibit protein synthesis via the ADP- ribosylation of the eukaryotic elongation factor (eEF2). Clear clinical benefit has been achieved in patients treated with an immunotoxin targeting CD22 expressed on B-cell malignancies and the immunotoxin has recently been approved by FDA. However, success has not been universal and, when treating solid tumors, immunotoxins have performed less well in producing complete responses, due to poor apoptotic responses. Screening for enhancing compounds revealed IAP inhibitors as potentially useful for improving immunotoxin killing. The immunotoxin, LMB-100, targets surface mesothelin on pancreatic tumor cells but when tested on two pancreatic adenocarcinoma cell lines, AsPC1 and BxPc3, there was no detectable reduction in cell viability. Upon further investigation, we found that birinapant, an IAP inhibitor, strongly synergized with this immunotoxin against AsPC1 but not against BxPC3 cells. We sought to understand the different responses in each cell line. Materials and Methods: viability was determined using the CellTiter-Glo Luminescent Cell Viability Assay kit (Promega, Madison WI). TNFalpha in the cell medium was detected using the hTNFa QuantiGlo ELISA (R&amp;D System). The level of the major cell-associated apoptotic proteins was assessed using the Human apoptosis Array kit (R&amp;D System). The spots on the arrays were quantified using ImageJ. Results: both AsPC1 and BxPC3 cells were resistant to the immunotoxin LMB-100. In the case of AsPC1, the immunotoxin caused only a modest cytostatic response at high immunotoxin concentrations at 72h whereas, in combination of birinapant, there was a strong cytotoxic effect, starting at 48h. BxPC3 cells remained resistant to the immunotoxin even in the presence of birinapant: the combination with the highest concentrations of both compounds reduced cell viability only to 50% after 72 hours. From the evaluation of the level of the major apoptotic proteins, we found that the most significative difference between the two cell lines was the phosphorylated status of p53, in particular the S392 hyper-phosphorylation in BxPC3 which has been associated with a poor prognosis, advanced tumor stage and grade in p53 positive cancers. AsPC1 cells also had a higher level of Bax, making them more predisposed to apoptosis. No difference in TNFalpha secreted in the medium or TNFR1 protein level was detected between the two cell lines. Conclusion: knowing the level of key proteins involved in the cell death pathway can explain the efficacy of drug combinations and could be used to predict responses. Citation Format: Antonella Antignani, David J FitzGerald. Synergy between IAP inhibitors and a cytotoxic antibody-based chimeric protein in pancreatic cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A018. doi:10.1158/1535-7163.TARG-19-A018

CK2 Inhibitor CX4945 Shows Strong In Vivo Anti Leukemia Effect in AML Via Augmented Ikaros-Mediated Regulation of Global Epigenetic Landscape

Over-expression of Casein Kinase II (CK2) pro-oncogenic kinase in Acute Myelogenous Leukemia (AML) is associated with poor prognosis. Inhibition of CK2 by siRNAs or with the specific inhibitor, CX-4945, shows strong cytotoxic effects. CK2 is a ubiquitous, constitutively active serine/ threonine kinase which has been implicated in leukemia progression via multiple mechanisms. One of the well-established mechanism is CK2 mediated phosphorylation and impairment of tumor suppressor function of Ikaros transcription factor. Impaired function of Ikaros has been associated with the development of leukemia including AML. In B-cell lymphoblastic leukemia, CK2 inhibition has been shown to exert a therapeutic effect via restoration and/or enhancement of the tumor suppressor activity of the Ikaros protein. The mechanism of therapeutic action of CK2 inhibition in AML is largely unknown and in vivo efficacy CK2 inhibitors in AML has never been established. Here we report anti-leukemia effect of CX-4945 in preclinical models of AML and demonstrate mechanism of action of CK2 inhibitor via restoration of Ikaros driven global regulation of epigenetic landscape. In order to study the effect of CK2 inhibition on Ikaros function in AML, we used U937 - a human myelomonocytic leukemia cell line with high baseline CK2 expression. U937 cells were treated with specific CK2 inhibitor, CX4945 for 72 hours. CK2 activity and Ikaros phosphorylation was measured using CK2 kinase assay and in vivo labeling - radio immunoblot assay. Results showed significant decrease in CK2 activity and Ikaros phosphorylation with no change in overall expression of the Ikaros protein. Expression analysis using RNA sequencing showed that treatment with CX-4945 caused significant upregulation of genes controlling immunity, and inflammation; and downregulation of genes involved in nucleic acid metabolism, RNA processing, and translation. Analysis of global genome-wide Ikaros occupancy using Chromatin Immunoprecipitation followed by next generation sequencing (ChIP-seq) of CX4945 treated U937 cells demonstrated that CX-4945 treatment significantly increased the number of Ikaros binding sites as well as increased peak strength while minimally re-distributing Ikaros' global genomic occupancy. While increased binding to the Promoter, Gene Body, and Gene Desert elements in CX-4945-treated cells was similar, the increase in Ikaros' DNA binding to enhancers was particularly pronounced. Further analysis showed that enhanced Ikaros DNA binding following treatment with CX-4945, directly induces formation of de novo enhancers. In order to test whether enhanced DNA binding of Ikaros is accompanied by augmented Ikaros function in the global epigenetic regulation of gene expression. We determined and compared chromatin accessibility of U937 cells before and after treatment with CX-4945, using ATAC-seq. Results indicate that Ikaros-induced de novo open chromatin at distal regulatory regions controls genes involved in the negative regulation of biological processes and cellular metabolism. Overall, these data demonstrate that, augmented Ikaros DNA-binding following CK2 inhibition resulted in 1) Ikaros' pioneering activity, 2) Ikaros' ability to induce the de novo formation of enhancers and super-enhancers, and 3) Ikaros' ability to induce the de novo formation of active enhancers and to activate poised enhancers. Together, these data uncover novel Ikaros functions in regulating the epigenetic landscape and identifies CK2 as a critical regulator of Ikaros activity. Next, we treated AML xenograft model of luciferase labelled U937 cells with CX4945 via oral gavage at dose 200mg/kg/day for 3 weeks and demonstrated significantly lower leukemia burden in treated group as measured by decreased bio-luminescence imaging. In summary, these results demonstrate for the first time that CK2 inhibitor, CX-4945 has strong anti-leukemia effect in AML preclinical models. One of the mechanisms by which CX4945 exert a therapeutic effect in AML involves enhancing Ikaros' function as regulator of global epigenomic landscape. These results provide strong mechanistic basis to develop novel targeted combination therapies using CK2 inhibitors for treatment of AML. Further studies evaluating combination therapies using patient derived xenograft (PDX) models of AML are underway. Disclosures Payne: Elf Zone, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

OPTIMAL FERMENTATION CONDITIONS FOR ANTIBIOTIC PRODUCTION BY ENDOPHYTIC Streptomyces cavourensis YBQ59 ISOLATED FROM Cinnamomum cassia Presl

This study determined the optimal nutrient and environmental conditions for the production of antimicrobial and cytotoxic agents by Streptomyces cavourensis YBQ59 during the fermentation process. The antimicrobial and cytotoxic activities of eluted fractions based gradient solvents via silica gel column of chromatograph were also evaluated. S. cavourensis YBQ59 exhibited strong, board-spectrum antimicrobial activities under fermentation conditions as follows: MT6 medium with soluble starch and soybean powder as carbon and nitrogen sources, respectively, temperature 30oC, initial pH 7.0, 20% DO concentration and with 5% initial seed culture. The kinetic of fermentation showed that the antimicrobial and cytotoxic activities were highest at between 72h and 78h. The fraction 25/1 D-M (dichloromethane –methanol) exhibited the highest antimicrobial effect against methicillin-resistant Staphylococcus aureus ATCC 33591 and methicillin-resistant Staphylococcus epidermidis ATCC 35984 with minimum inhibitory concentrations of 9.4 µg/ml and 6.4 µg/ml, respectively. The fraction 10/1 D-M had strong cytotoxic effects towards multidrug-resistant A459 and H1299 lung carcinoma cell lines with the cell viability of 11.3% and 12.4%, respectively. In conclusion, S. cavourensis YBQ59 would be a potential producer of valuable bioactive compounds that may have a board application in pharmaceutical industry and agriculture (biocontrol, livestock, food safety and quality management)