Pro-apoptotic Activity Research Articles

Integrating a quinone substructure into histone deacetylase inhibitors to cope with Alzheimer's disease and cancer.

Alzheimer's disease (AD) and cancer are among the most devastating diseases of the 21st century. Although the clinical manifestations are different and the cellular mechanisms underlying the pathologies are opposite, there are different classes of molecules that are effective in both diseases, such as quinone-based compounds and histone deacetylase inhibitors (HDACIs). Herein, we investigate the biological effects of a series of compounds built to exploit the beneficial effects of quinones and histone deacetylase inhibition (compounds 1-8). Among the different compounds, compound 6 turned out to be a potent cytotoxic agent in SH-SY5Y cancer cell line, with a half maximal inhibitory concentration (IC50) value lower than vorinostat and a pro-apoptotic activity. On the other hand, compound 8 was nontoxic up to the concentration of 100 μM and was highly effective in stimulating the proliferation of neural precursor cells (NPCs), as well as inducing differentiation into neurons, at low micromolar concentrations. In particular, it was able to induce NPC differentiation solely towards a neuronal-specific phenotype, without affecting glial cells commitment.

Development of self-nanoemulsifying drug delivery system of 1-(2,5-dihydroxy phenyl)-3-pyridine-2-yl-propenone to enhance pro-apoptotic activities in MCF-7 breast cancer cells

Development of self-nanoemulsifying drug delivery system of 1-(2,5-dihydroxy phenyl)-3-pyridine-2-yl-propenone to enhance pro-apoptotic activities in MCF-7 breast cancer cells

In vitro pro-apoptotic and anti-metastatic potentials of harmaline-silver containing folate-linked chitosan nano-drug delivery system.

Harmaline and green-synthesized silver nanoparticles were encapsulated by folate-linked chitosan molecules as a receptor-mediated drug delivery system to evaluate its pro-apoptotic and anti-metastatic potentials on human ovarian (A2780) and epithelioid (PANC) cancer cells. The Ag nanoparticles (AgNP) were synthesized utilizing an herbal bio-platform (Bistorta officinalis) and embedded with harmalin. The Harmaline-ag containing folate-linked chitosan nanoparticles (HA-fCNP) were synthesized utilizing the ionic gelation method. Both the AgNP and HA-fCNP nanoparticles were characterized by DLS, FESEM, and Zeta potential analysis. Moreover, the chemical properties of HA-fCNP and the crystallinity of AgNPs were determined by applying FTIR and XRD methods, respectively. The HA-fCNP cytotoxicity was analyzed on A2780, PANC, and HFF cell lines. Moreover, pro-apoptotic and anti-metastatic potentials of HA-fCNP were studied by analyzing the BAX-BCL2 and MMP2-MMP9 gene expression profiles, respectively. The A2780 cellular death was determined by AO/PI and flow cytometry methods. The HA-fCNP significantly exhibited a selective cytotoxic impact on A2780 and PANC cancerous cell lines compared with normal human foreskin fibroblast (HFF) cells. The increased SubG1-arrested A2780 cells and up-regulated BAX gene expression following the increased treatment concentrations of hA-fCNP indicated its selective pro-apoptotic activity on A2780 cells. Also, the notable down-regulated expressions of MMP2 and MMP9 metastatic genes following the increasing doses of HA-fCNP treatment on A2780 cells confirmed its anti-metastatic activity. The cancer-selective cytotoxicity, apoptotic, and anti-metastatic properties of HA-fCNP are considered the appropriate properties of an anticancer compound.

Biological Activities of Molecules Derived from Olea europaea L. Tested In Vitro.

Extra virgin olive oil is a typical food of the Mediterranean area, obtained by pressing Olea europaea L. fruits. Its polyphenols have been studied for their antioxidant function and protective action against cancer and chronic kidney disease. In this in vitro study, we tested titrated extracts from Olea europaea L. on a human embryonic kidney 293 (HEK-293E) cell line, regarding their pro-apoptotic and antiproliferative capacities, using " IncuCyte® S3 Live-Cell Analysis System". We selected Olea europaea L. active compounds like hydroxytyrosol (HT) and oleuropein (OLE). These extracts were tested at different concentrations and characterized by HPLC-DAD-MS for the content in secondary active metabolites. The real-time observation of cell behavior was performed by IncuCyte, which can quantitatively analyze the cell proliferation and death. This study showed that all the tested extracts can significantly inhibit cellular growth at 50 µM but the reduced proliferation is not related to an increase in cellular apoptosis. Instead, the same analysis performed by using extracts at 100 µM reveals that they can inhibit cellular growth, further inducing cellular apoptosis. The results on the HEK-293E cells confirmed the antiproliferative and proapoptotic actions of active compounds from an Olea europaea L. matrix in this cell line.

Scoparone from Artemisia capillaris Thunb. induces apoptosis in HepG2 cells via activation of both intracellular and extracellular pathways

Six separated compounds were identified from Artemisia capillaris Thunb., and they were 7-methoxycoumarin (1), 6,7-dimethoxycoumarin (2), 7-hydroxy-6-methoxycoumarin (3), quercetin (4), chlorogenic acid (5) and caffeic acid (6). Among them, 6,7-dimethoxycoumarin, as known as scoparone, was the most effective on scavenging ABTS free radicals (IC50 = 0.97 μΜ) and was then tested by cytotoxic activity and pro-apoptotic activity against HepG2 cells. Scoparone dose-dependently and time-dependently inhibited the cell proliferation. Furthermore, scoparone induced the expression of Bax, concurrently suppressing the expression of Bcl-2, resulting in a noteworthy elevation in the Bax/Bcl-2 ratio to up-regulate Caspase-3 activity, thus inducing cell apoptosis via the intracellular pathway. Meanwhile, scoparone promoted the expression of Fas, FasL, FADD, Caspase-8 and Caspase-3, indicating that scoparone also triggered apoptosis via the extracellular pathway. In a word, scoparone demonstrated remarkable antitumor capability to induce apoptosis of HepG2 cells through both intracellular and extracellular pathways.

Liraglutide attenuates obese-associated breast cancer cell proliferation via inhibiting PI3K/Akt/mTOR signaling pathway

This study aims to explore the anti-proliferative, pro-apoptotic, and anti-migration activities of liraglutide (LGT) in MCF-7 breast cancer (BC) cells in subjects with obesity, particularly its effects on the PI3K/Akt/mTOR/AMPK pathway. The role of AMPK/SIRT-1, an essential regulator of adipokine production, in the effect of LGT on the production of adipose-derived adipokine was also assessed. MCF-7 cells were incubated in conditioned medium (CM) generated from adipose-derived stem cells (ADSCs) of obese subjects. MCF-7 cells were then treated with LGT for 72 h. Anti-proliferative, pro-apoptotic, and anti-migration activities were investigated using alamarBlue, annexin V stain, and scratch assay, respectively. Protein levels of phosphorylated PI3K, p-Akt, p-mTOR, and p-AMPK were investigated using immunoblotting. Levels of adipokines in ADSCs were determined using RT-PCR before and after transfection of ADSCs using the specific small interference RNA sequences for AMPK and SIRT-1. LGT evoked anti-proliferative, apoptotic, and potential anti-migratory properties on MCF-7 cells incubated in CM from obese ADSCs and significantly mitigated the activity of the PI3K/Akt/mTOR survival pathway—but not AMPK—in MCF-7 cells. Furthermore, the anti-proliferative effects afforded by LGT were similar to those mediated by LY294002 (PI3K inhibitor) and rapamycin (mTOR inhibitor). Our results reveal that transfection of AMPK/SIRT-1 genes did not affect the beneficial role of LGT in the expression of adipokines in ADSCs. In conclusion, LGT elicits anti-proliferative, apoptotic, and anti-migratory effects on BC cells in obese conditions by suppressing the activity of survival pathways; however, this effect is independent of the AMPK/SIRT1 pathway in ADSCs or AMPK in BC cells.

Transforming growth factors β and their signaling pathway in renal cell carcinoma and peritumoral space—transcriptome analysis

PurposeThe aim of the study was to verify hypotheses: Are transforming growth factors TGFβ1-3, their receptors TGFβI-III, and intracellular messenger proteins Smad1-7 involved in the pathogenesis of kidney cancer? What is the expression of genes of the TGFβ/Smads pathway in renal cell carcinoma (RCC) tissues, peritumoral tissues (TME; tumor microenvironment), and in normal kidney (NK) tissue?.MethodsTwenty patients with RCC who underwent total nephrectomy were included into the molecular analysis. The mRNA expression of the genes was quantified by RT-qPCR.ResultsThe study showed that the expression of the genes of TGFβ/Smads pathway is dysregulated in both RCC and the TME: TGFβ1, TGFβ3 expression is increased in the TME in comparison to the NK tissues; TGFβ2, TGFβ3, TGFβRI, TGFβRIII, Smad1, Smad2, Smad3, and Smad6 are underexpressed in RCC comparing to the TME tissues; TGFβRI, TGFβRIII, and Smad2 are underexpressed in RCC in comparison to the NK tissues.ConclusionOn the one hand, the underexpression of the TGFβ signaling pathway genes within the malignant tumor may result in the loss of the antiproliferative and pro-apoptotic activity of this cytokine. On the other hand, the overexpression of the TGFβ/Smads pathway genes in the TME than in tumor or NK tissues most probably results in an immunosuppressive effect in the space surrounding the tumor and may have an antiproliferative and pro-apoptotic effect on non-neoplastic cells present in the TME. The functional and morphological consistency of this area may determine the aggressiveness of the tumor and the time in which the neoplastic process will spread.

BRAF and MEK inhibitor combinations induce potent molecular and immunological effects in NRAS-mutant melanoma cells: Insights into mode of action and resistance mechanisms.

About 25% of melanoma harbor activating NRAS mutations, which are associated with aggressive disease therefore requiring a rapid antitumor intervention. However, no efficient targeted therapy options are currently available for patients with NRAS-mutant melanoma. MEK inhibitors (MEKi) appear to display a moderate antitumor activity and also immunological effects in NRAS-mutant melanoma, providing an ideal backbone for combination treatments. In our study, the MEKi binimetinib, cobimetinib and trametinib combined with the BRAF inhibitors (BRAFi) encorafenib, vemurafenib and dabrafenib were investigated for their ability to inhibit proliferation, induce apoptosis and alter the expression of immune modulatory molecules in sensitive NRAS-mutant melanoma cells using two- and three-dimensional cell culture models as well as RNA sequencing analyses. Furthermore, NRAS-mutant melanoma cells resistant to the three BRAFi/MEKi combinations were established to characterize the mechanisms contributing to their resistance. All BRAFi induced a stress response in the sensitive NRAS-mutant melanoma cells thereby significantly enhancing the antiproliferative and proapoptotic activity of the MEKi analyzed. Furthermore, BRAFi/MEKi combinations upregulated immune relevant molecules, such as ICOS-L, components of antigen-presenting machinery and the "don't eat me signal" molecule CD47 in the melanoma cells. The BRAFi/MEKi-resistant, NRAS-mutant melanoma cells counteracted the molecular and immunological effects of BRAFi/MEKi by upregulating downstream mitogen-activated protein kinase pathway molecules, inhibiting apoptosis and promoting immune escape mechanisms. Together, our study reveals potent molecular and immunological effects of BRAFi/MEKi in sensitive NRAS-mutant melanoma cells that may be exploited in new combinational treatment strategies for patients with NRAS-mutant melanoma.

Design, synthesis and bioactivity evaluation of novel fusion peptides and their CPT conjugates inducing effective anti-tumor responses on HER2 positive tumors

Human epidermal growth factor receptor 2 (HER2) represents an ideal target for antibody drug development, abnormal expression of the HER2 gene is associated with multiple tumor types. Pertuzumab, as the first monoclonal antibody inhibitor of HER2 dimerization, has been FDA-approved for HER2-positive patients. In order to enhance the activity of HER2-targeted peptide-drug conjugates (PDCs) developed based on pertuzumab, a novel class of conjugates 1–9 was designed and synthesized by fusing the N-terminal peptide sequence of the second mitochondria-derived activator of caspases (SMAC) with P1, followed by conjugation with CPT molecules. Compound 4 exhibited excellent in vitro anti-tumor activity across the three HER2-positive cell lines, comparable to the activity of CPT. Apoptosis induction assays indicated that the synergistic effect of the SMAC sequence enhanced the pro-apoptotic activity of the conjugate. Western Blot analysis and Caspase activity studies validated the mechanism through which SMAC peptides, in synergy with CPT, enhance the activity of PDCs. In vivo studies demonstrated that compound 4 possesses superior anti-tumor activity compared to CPT and can effectively mitigate potential renal toxicity associated with free SMAC peptides. In conclusion, conjugate 4 exhibited excellent anti-tumor activity both in vitro and in vivo, offering potential for further development as a novel peptide-conjugated drug.

Hellebrigenin induces oral cancer cell apoptosis by modulating MAPK signalling and XIAP expression.

Oral squamous cell carcinoma (OSCC), which accounts for 90% of all oral cancers, has become a public health crisis worldwide. despite advances in therapeutic interventions, the prognosis remains poor for advanced-stage OSCC. In this study, we investigate the anticancer activity and the mode of action of hellebrigenin in human OSCC. The findings demonstrated that hellebrigenin exerted cytotoxic effects in OSCC cells through cell cycle arrest at the G2/M phase and downregulation of cell cycle-related proteins (cyclins A2, B1 and D3, Cdc2, CDK4 and CDK6). Moreover, hellebrigenin caused activation of PARP and caspase 3, 8 and 9, followed by downregulation of antiapoptotic proteins (Bcl-2 and Bcl-xL) and upregulation of pro-apoptotic proteins (Bax and Bak). The hellebrigenin treatment also increased Fas, DR5, DcR2 and DcR3 expressions in oral cancer cells, indicating the compound causes oral cancer cell apoptosis through both intrinsic and extrinsic pathways. Regarding upstream signalling, hellebrigenin was found to reduce the phosphorylation of ERK, p38, and JNK, indicating that hellebrigenin triggers caspase-mediated apoptosis by downregulating MAPK signalling pathway. Finally, the human apoptosis array findings revealed that hellebrigenin specifically suppressed the expression of XIAP to execute its pro-apoptotic activities. Taken together, the study suggests that hellebrigenin can act as a potent anticancer compound in human OSCC.

In Vitro Evidence of Selective Pro-Apoptotic Action of the Pure Cannabidiol and Cannabidiol-Rich Extract

Plant cannabinoids, secondary metabolites of species belonging to the Cannabis genus, can mimic the endocannabinoids’ action and exert biological effects. Considering the contribution of the endocannabinoid system in cell cycle and apoptotic regulation, there is an interest in exploring the potential anti-cancer activities of natural and synthetic cannabinoids. Cannabidiol (CBD), an abundant plant cannabinoid, reveals a low affinity to cannabinoid receptors and, contrary to various cannabinoids, lacks psychoactive action. Here, we present the in vitro assessment of the pro-apoptototic potential of CBD-rich extracts of Cannabis sativa L. (eCBD) compared to purified CBD (pCBD). As demonstrated, both eCBD and pCBD decreased the viability of breast cancer cell line MDA-MB-231 and human prostate cancer cell line PC-3 in a concentration-dependent fashion. Endoplasmic reticulum stress-related apoptosis and morphological changes were induced only in low-serum conditions. Moreover, the effects of eCDB and pCDB were also assessed in non-malignant cell lines (MCF-10A and PNT2) with no alterations of viability noted, ultimately suggesting a selective action of CBD in tumor cells. The results suggest the possible involvement of reactive oxygen species in the response mechanism to eCBD and pCBD, but no clear pattern was observed. We also demonstrated significant changes in gene expression involved in apoptosis and cell cycle control upon extract treatment. Altogether, our study shows the potential of eCBD and pCBD as novel pro-apoptototic agents that can be considered promising in future preclinical and clinical testing.

Melanocortin receptor 4 as a new target in melanoma therapy: Anticancer activity of the inhibitor ML00253764 alone and in association with B-raf inhibitor vemurafenib

The aim of our study is to investigate in vitro and in vivo MC4R as a novel target in melanoma using the selective antagonist ML00253764 (ML) alone and in combination with vemurafenib, a B-rafV600E inhibitor.The human melanoma B-raf mutated A-2058 and WM 266-4 cell lines were used. An MC4R null A-2058 cell line was generated using a CRISPR/Cas9 system. MC4R protein expression was analysed by western blotting, immunohistochemistry, and immunofluorescence. Proliferation and apoptotic assays were performed with ML00253764, whereas the synergism with vemurafenib was evaluated by the combination index (CI) and Loewe methods. ERK1/2 phosphorylation and BCL-XL expression were quantified by western blot. In vivo experiments were performed in Athymic Nude-Foxn1nu male mice, injecting subcutaneously melanoma cells, and treating animals with ML, vemurafenib and their concomitant combination. Comet and cytome assays were performed.Our results show that human melanoma cell lines A-2058 and WM 266-4, and melanoma human tissue, express functional MC4R receptors on their surface. MC4R receptors on melanoma cells can be inhibited by the selective antagonist ML, causing antiproliferative and proapoptotic activity through the inhibition of phosphorylation of ERK1/2 and a reduction of BCL-XL. The concomitant combination of vemurafenib and ML caused a synergistic effect on melanoma cells in vitro and inhibited in vivo tumor growth in a preclinical model, without causing mouse weight loss or genotoxicity.Our original research contributes to the landscape of pharmacological treatments for melanoma, providing MC4R antagonists as drugs that can be added to established therapies.

Discovery of a Novel, First-in-Class Bfl-1 BH3 Mimetic with Pro-Apoptotic Activity

Background: Intrinsic apoptosis is regulated by the B Cell lymphoma 2 (BCL-2) protein family. Anti-apoptotic family members, such as BCL-2, MCL-1 and BFL-1, sequester their pro-apoptotic counterparts through a highly conserved BH3 binding groove. Disruption of this balance leads to mitochondrial pore formation and subsequent cell death. To evade apoptosis, tumors often upregulate the expression of one or more anti-apoptotic family members and as such they have become attractive targets for anti-cancer drug development. BFL-1 (‘Bcl-2 related gene expressed in fetal liver’, gene name BCL2A1) is a lesser-known anti-apoptotic family member, physiologically mainly expressed in the hematopoietic system. BCL2A1 overexpression has been reported in many types of B cell lymphoma. Diffuse Large B Cell Lymphoma (DLBCL) patient sample analysis showed that BCL2A1 expression levels are second only to MCL1 within the anti-apoptotic family. Expression of BFL-1 was shown to be upregulated in MYC+/BCL2+ double hit lymphoma cell lines treated with the BCL-2 inhibitor, Venetoclax, in vivo. Interestingly, BFL-1 positive lymphomas are less sensitive to inhibition of BCL-2 and MCL-1. Hence, targeting BFL-1 is a rational and attractive strategy in DLBCL to improve clinical outcome. Methods: Using hit identification screens followed by iterative structure-activity relationship (SAR) analyses and structure-based design, compound A, a first-in-class, selective BFL-1 BH3 mimetic, was identified. Compound A was evaluated using biochemical, cellular in vitro and in vivo tumor efficacy models. Results: Compound A is a highly potent and selective compound in biochemical assays. In BFL-1 sensitive lymphoma cell lines characterized by high BFL-1 protein expression, compound A was shown to displace BAK and BIM from BFL-1, leading to downstream cleavage of caspase 3 and cell death. Interestingly, these cellular models were resistant to Venetoclax. Compound A exhibited tumor growth inhibition in an engineered BFL-1 overexpressing, MCL-1 KO human multiple myeloma xenograft model. However, in the high BFL-1 expressing SU-DHL-2 DLBCL human xenograft model, compound A did not induce significant PD (cleaved caspase 3) and efficacy in vivo. Conclusion: First-in-class selective BFL-1 BH3 mimetics are shown to specifically inhibit cell growth of lymphoma cell lines in vitro and can induce efficacy in vivo in a BFL-1 overexpressing model. Further studies may be useful to determine whether compound A elicits anti-tumor efficacy in lymphoma alone or in combination with other anti-apoptotic agents.

The PP2A-B56α Heterocomplex Regulates Response to Venetoclax Plus Azacitidine Treatment in AML

Venetoclax (VEN), a selective inhibitor of BCL2, combined with azacitidine (Aza) has emerged as the new standard of care for patients with acute myeloid leukemia (AML) ineligible for intensive chemotherapy. However, the long-term medical benefit is modest, and relapse appears to be unavoidable. We have recently reported that PP2A activators enhance the efficacy of VEN and VEN+Aza in primary samples and pre-clinical models of AML, and that the PP2A-B56α complex drives the synergistic pro-apoptotic activity observed. Our aim here is to further decipher the role of PP2A-B56α in VEN+Aza treatment response in AML. We used CRISPR-Cas9 to generate HL-60 sublines lacking the B56α or B55α PP2A regulatory subunits. Interestingly, while there were no significant differences in their baseline rate of proliferation, HL-60 B56α knockdown (KD) clones were significantly more resistant to VEN and VEN+Aza than wild type (WT) cells. In contrast, HL-60 B55α KD clones were sensitive to VEN+Aza, pointing out a specific role for specific PP2A heterotrimers, in this case the PP2A-B56α complex in the combined treatment response. Protein expression analysis of known substrates of PP2A-B56α demonstrated that pSer62-MYC and total MYC decreased after VEN+Aza treatment in WT cells but not in KD cells. It is known that the PP2A-B56α complex directly dephosphorylates MYC-Ser62, inducing MYC ubiquitin-mediated proteasomal degradation. Moreover, p21/CDKN1A, that is transcriptionally activated by p53 and inhibited by MYC, was up-regulated at both the mRNA and protein level after VEN+Aza treatment in WT cells but not in KD cells. HL-60 is a p53 null cell line; therefore, p21 up-regulation upon the combined treatment might be MYC-dependent. Of note, treatment of HL-60 B56α KD cells with the MYC inhibitor MYCi975 rescued the VEN+Aza synergy seen in these cells, confirming the relevance of MYC degradation to VEN+Aza response. Interestingly, VEN+MYCi975 had synergistic anti-leukemic effects in these same cell lines. Furthermore, CDKN1A expression was also rescued upon the triple therapy consisting of VEN+Aza+MYCi975. Combined, our results suggest that the PP2A-B56α complex is involved in the regulation of VEN+Aza response through changes in MYC expression. Interestingly, a novel small molecular glue (PMG) developed by our group, which specifically stabilizes the PP2A-B56α complex, enhanced VEN plus Aza response in vitro; however, this triple therapy did not work in HL-60 B56α KD cells and we observed a clearly decrease in MYC protein expression upon the triple therapy only in HL-60 B56α WT cells, supporting the rationale to translate these novel PMGs into the clinic. Altogether, our data suggest that PP2A-B56α might have an important role in VEN plus Aza treatment response through the regulation of MYC degradation.

Potency and Efficacy of Pharmacological PIP4K2 Inhibitors in Acute Lymphoblastic Leukemia

Introduction and aims: Adult acute lymphoblastic leukemia (ALL) is a heterogeneous group of highly aggressive hematological malignancies with high recurrence rates and poor prognosis. Pharmacological inhibition of proteins of the phosphatidylinositol-5-phosphate 4-kinase type 2 family (PIP4K2s), through the compound THZ-P1-2, exhibits antileukemic activity by interrupting mitochondrial homeostasis and autophagy. Nevertheless, different pharmacological inhibitors with distinct affinities for PIP4K2s have been developed (PIP4K-IN-a131 and CC260), but their potential as antileukemic agents has been little explored. In the present study, we investigated the expression of PIP4K2s in ALL patients and cell models. Furthermore, the potency and efficacy of three PIP4K2s inhibitors were characterized in ALL cells. Material and methods: The gene expression of PIP4K2A, PIP4K2B and PIP4K2C was evaluated in a cohort composed of 12 healthy donors and 101 patients with ALL by quantitative PCR. Gene and protein expression of PIP4K2s was investigated in a panel containing nine ALL cell lines. The potency and efficiency of three pharmacological inhibitors of PIP4K2s (THZ-P1-2, PIP4K-IN-a131, and CC260) were investigated in cell lines (Jurkat, MOLT4, NALM6, and Namalwa) and primary cells from patients with ALL. Cell viability was assessed by MTT assay, annexin V/PI labeling and mitochondrial membrane potential by flow cytometry, and proteins involved in cell signaling by Western blotting. Comparisons were performed by ANOVA and Bonferroni post-test and a p-value < 0.05 was considered significant. Results: PIP4K2A mRNA levels were higher in ALL patients ( p < 0.05), whereas PIP4K2B levels were lower ( p < 0.01) compared to healthy controls. PIP4K2C expression did not differ between groups. Gene and protein expression of PIP4K2s showed a good correlation, and the expression of PIP4K2A and PIP4K2C showed the greatest variations between ALL cell lines. Pharmacological inhibitors of PIP4K2s showed reduced viability dependent on time and concentration, with THZ-P1-2 being the one with the highest pro-apoptotic activity (IC 50 range: 1.4 - 8.1 µM), followed by PIP4K-IN-a131 (1.4 - >50 µM) and CC260 (13.3 - >50 µM). Similar results were observed in primary cells from patients with ALL. THZ-P1-2 also showed better efficacy in inducing apoptosis and mitochondrial damage compared to PIP4K-IN-a131 and CC260 ( p < 0.05). From a molecular point of view, both THZ-P1-2 and PIP4K-IN-a131 similarly induced markers of apoptosis (PARP1 cleavage) and DNA damage (γH2AX). However, only THZ-P1-2 induced markers of blocking autophagic outflow (increased LC3BII and SQSTM1/p62) and reduced RPS6 phosphorylation (a target of the PI3K/AKT/mTOR pathway). Discussion and Conclusion: A differential expression PIP4K2A and PIP4K2C gene expression profile was observed in a cohort of patients with ALL. THZ-P1-2 was the PIP4K2 inhibitor with the highest anti-leukemic activity and exerted its effects through a dose-dependent apoptosis induction and modulation of the autophagic process. An analysis of the structure-activity of the compounds may help to identify the most promising pharmacophores groups and promote advances in the optimization of this potential class of antileukemic agents. Support by FAPESP, CNPq and CAPES.

Chemical profile, antiproliferative and pro-apoptotic activities of essential oils of Pulicaria arabica against A549 lung cancer cell line

Pulicaria arabica has been traditionally utilized in folk medicine for various purposes such as ulcer treatments as well as antidiarrheal agent. Herein, the chemical profiles of Pulicaria arabica essential oils (PAEOs) and the in vitro antiproliferative effect of PAEOs were investigated. Hydrodistillation was employed to prepare PAEOs which were then characterized by GC/MS, while the antiproliferative effects were investigated by MTT assay as well as flow cytometric and RT-PCR analysis. Sixty-four (99.99 %) constituents were recognized from PAEOs. Carvotanacetone (36.97 %), (-)-carvomenthone (27.20 %) and benzene, 2-(1,1-dimethylethyl)-1,4-dimethoxy- (6.92 %) were the main components. PAEOs displayed IC50 values ranging from 30 to 50 μg/mL. DNA content analysis revealed that A549 cells exposed to PAEOs exhibited an increase in G1 cells population. The flow cytometry analysis results also showed that the PAEOs antiproliferative effect was mediated via apoptosis induction. Furthermore, a modulation in the pro-apoptotic markers (caspase-3 and Bax) and anti-apoptotic (Bcl-2) was also observed. In conclusion, PAEOs exhibited a moderate anti-proliferative effect on A549 cells through modulating the cell cycle progression and apoptosis initiation. These findings could offer a potential therapeutic use of PAEOs in lung cancer treatment.

Phenol-Soluble Modulin α3 Stimulates Autophagy in HaCaT Keratinocytes.

Phenol-soluble modulins (PSMs) are pore-forming toxins (PFTs) produced by staphylococci. PSMs exert diverse cellular effects, including lytic, pro-apoptotic, pro-inflammatory and antimicrobial actions. Since the effects of PSMs on autophagy have not yet been reported, we evaluated the autophagic activity in HaCaT keratinocytes treated with recombinant PSMα3. The autophagic flux and levels of autophagic marker proteins were determined using Western blot analysis. Subcellular localization of LC3B and Beclin-1 was investigated using an indirect immunofluorescence assay. The ultrastructural features of control and PSMα3-treated cells were evaluated via transmission electron microscopy. Cytoplasmic acidification was measured via acridine orange staining. Phosphorylation levels of protein kinases, implicated in autophagy regulation, were studied using a phospho-kinase array and Western blot analysis. PSMα3 facilitated the intracellular redistribution of LC3B, increased the average number of autophagosomes per cell, promoted the development of acidic vesicular organelles, elevated the levels of LC3B-II, stimulated autophagic flux and triggered a significant decrease in the net autophagic turnover rate. PSMα3 induced the accumulation of autophagosomes/autolysosomes, amphisomes and multilamellar bodies at the 0.5, 6 and 24 h time points, respectively. The phospho-Akt1/2/3 (T308 and S473), and phospho-mTOR (S2448) levels were decreased, whereas the phospho-Erk1/2 (T202/Y204 and T185/Y187) level was increased in PSMα3-treated cells. In HaCaT keratinocytes, PSMα3 stimulates autophagy. The increased autophagic activity elicited by sub-lytic PSM concentrations might be an integral part of the cellular defense mechanisms protecting skin homeostasis.

Unraveling the Mechanisms of Sensitivity to Anti-FGF Therapies in Imatinib-Resistant Gastrointestinal Stromal Tumors (GIST) Lacking Secondary KIT Mutations.

We showed previously that inhibition of KIT signaling in GISTs activates FGFR-signaling pathway rendering cancer cells resistant to receptor tyrosine kinase inhibitor (RTKi) imatinib mesylate (IM) (Gleevec) despite of absence of secondary KIT mutations and thereby illustrating a rationale for the combined (e.g., KIT- and FGFR-targeted) therapies. We show here that long-term culture of IM-resistant GISTs (GIST-R1) with IM substantially down-regulates KIT expression and induces activation of the FGFR-signaling cascade, evidenced by increased expression of total and phosphorylated forms of FGFR1 and 2, FGF-2, and FRS-2, the well-known adaptor protein of the FGF-signaling cascade. This resulted in activation of both AKT- and MAPK-signaling pathways shown on mRNA and protein levels, and rendered cancer cells highly sensitive to pan-FGFR-inhibitors (BGJ 398, AZD 4547, and TAS-120). Indeed, we observed a significant decrease of IC50 values for BGJ 398 in the GIST subclone (GIST-R2) derived from GIST-R1 cells continuously treated with IM for up to 12 months. An increased sensitivity of GIST-R2 cells to FGFR inhibition was also revealed on the xenograft models, illustrating a substantial (>70%) decrease in tumor size in BGJ 398-treated animals when treated with this pan-FGFR inhibitor. Similarly, an increased intra-tumoral apoptosis as detected by immunohistochemical (IHC)-staining for cleaved caspase-3 on day 5 of the treatment was found. As expected, both BGJ 398 and IM used alone lacked the pro-apoptotic and growth-inhibitory activities on GIST-R1 xenografts, thereby revealing their resistance to these TKis when used alone. Important, the knockdown of FGFR2, and, in much less content, FGF-2, abrogated BGJ 398's activity against GIST-R2 cells both in vitro and in vivo, thereby illustrating the FGF-2/FGFR2-signaling axis in IM-resistant GISTs as a primary molecular target for this RTKi. Collectively, our data illustrates that continuous inhibition of KIT signaling in IM-resistant GISTs lacking secondary KIT mutations induced clonal heterogeneity of GISTs and resulted in accumulation of cancer cells with overexpressed FGF-2 and FGFR1/2, thereby leading to activation of FGFR-signaling. This in turn rendered these cells extremely sensitive to the pan-FGFR inhibitors used in combination with IM, or even alone, and suggests a rationale to re-evaluate the effectiveness of FGFR-inhibitors in order to improve the second-line therapeutic strategies for selected subgroups of GIST patients (e.g., IM-resistant GISTs lacking secondary KIT mutations and exhibiting the activation of the FGFR-signaling pathway).

Pro-Apoptotic Activity of Epi-Obtusane against Cervical Cancer: Nano Formulation, In Silico Molecular Docking, and Pharmacological Network Analysis.

Cancer is a major disease that threatens human health all over the world. Intervention and prevention in premalignant processes are successful ways to prevent cancer from striking. On the other hand, the marine ecosystem is a treasure storehouse of promising bioactive metabolites. The use of such marine products can be optimized by selecting a suitable nanocarrier. Therefore, epi-obtusane, previously isolated from Aplysia oculifera, was investigated for its potential anticancer effects toward cervical cancer through a series of in vitro assays in HeLa cells using the MTT assay method. Additionally, the sesquiterpene was encapsulated within a liposomal formulation (size = 130.8 ± 50.3, PDI = 0.462, zeta potential -12.3 ± 2.3), and the antiproliferative potential of epi-obtusane was investigated against the human cervical cancer cell line HeLa before and after encapsulation with liposomes. Epi-obtusane exhibited a potent effect against the HeLa cell line, while the formulated molecule with liposomes increased the in vitro antiproliferative activity. Additionally, cell cycle arrest analysis, as well as the apoptosis assay, performed via FITC-Annexin-V/propidium iodide double staining (flow cytofluorimetry), were carried out. The pharmacological network enabled us to deliver further insights into the mechanism of epi-obtusane, suggesting that STAT3 might be targeted by the compound. Moreover, molecular docking showed a comparable binding score of the isolated compound towards the STAT3 SH2 domain. The targets possess an anticancer effect through the endometrial cancer pathway, regulation of DNA templated transcription, and nitric oxide synthase, as mentioned by the KEGG and ShinyGo 7.1 databases.

A New Class of Synthetic Type-B Polycyclic Polyprenylated Acylphloroglucinols (PPAP-B) Overcomes Venetoclax-Resistance in Acute Myeloid Leukemia (AML)

Venetoclax (VEN) resistance in AML therapy is a field of unmet need. VEN selectively binds in the BH3-binding groove of BCL-2 to release BIM leading to pore-forming BAX/BAK dimerization to finally release cytochrome C, as the “step of no return” in induction of programmed cell death. Compensatory overexpression of other BCL-2 family members such as MCL-1 and BCL-XL is a well described resistance mechanism to restore the ability to sequester BIM and therefore compensate for BCL-2 inhibition. Type-B PPAP are a novel class of neoplastic therapeutics. A new synthetic toolbox for the coupling of a bioactive head group (R 3) enabled the generation of novel, highly active PPAPs, which are low-toxic, water-soluble and photochemically stable - and therefore attractive compounds for clinical development. We now provide evidence that PPAP-B possess antileukemic activity in in vitro and ex vivo AML models via induction of the mitochondrial apoptosis pathways. Further, PPAP-B suppress transcription of MCL-1 and BCL-XL - leading to highly synergistic induction of apoptosis in combination with venetoclax, including a Venetoclax-resistant AML model. Employing structure-activity relationship (SAR) studies, newly generated PPAPs (#22-#144) were tested in acute leukemia HL60, Molm14 and OCI-AML-3 cell lines as well as freshly harvested native leukemia blasts from consented patients. Physiologic mononuclear cells (MC) extracted from cord blood or healthy donors were used as controls. PPAPs were modified step-by-step regarding their chemical structure and assessed in dose dilution series to determine antileukemic efficacy of the compounds. The capability to induce apoptosis and inhibit proliferation was assessed, using standard techniques. To shed light on the underlying mechanism of action, Proteome Profiler Arrays (R&D systems) were performed and confocal microscopy was employed to trace specific fluorescent PPAPs within the cell. Together, the SAR approach led to improvement of IC50 rates of PPAP-B ranging from 20 μM to now < 4 μM. Compound #122 was thereby identified as the most potent PPAP with respect to inhibition of proliferation and induction of apoptosis. Genotoxic studies, as evidenced in a thymidine kinase mouse lymphoma assay (MLA), did not point to potential genotoxicity. Confocal microscopy confirmed accumulation of PPAPs in the cytosol and outer mitochondrial layer using fluorescent PPAPs. In line, induction of mitochondrial apoptosis was seen as evidenced by BAX activation, release of cytochrome C and cleavage of caspases 8 and 3. Further, PPAP-B suppressed transcription of MCL-1 and BCL-XL, putatively via inhibition of the JAK-STAT pathway as discussed earlier. These observations suggest a role of PPAPs in overcoming VEN-resistance in AML. Indeed, combination of PPAP #122 with VEN was highly synergistic with regard to induction of apoptosis, already at doses where the single agents did not display pro-apoptotic activity (e.g. PPAP #122 2.5 μM: 10%, VEN 5nM: 5%, PPAP/VEN combination:74% apoptotis). Most interestingly, PPAP #122 displayed strong pro-apoptotic activity in VEN-resistant leukemia models (MOLM14, HL60, OCi-3 models) with a IC50s < 1μM, and in combination with VEN, re-sensitized cells towards Venetoclax in a combination approach. Together, further development of type-B PPAPs to target Venetoclax resistance in AML is a promising approach to overcome a so far unmet clinical need in acute leukemias.