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Cisplatin, which induces DNA damage, is standard chemotherapy for advanced bladder cancer (BCa). However, efficacy is limited due to resistance development. Since artesunate (ART), a derivative of artemisinin originating from Traditional Chinese Medicine, has been shown to exhibit anti-tumor activity, and to inhibit DNA damage repair, the impact of artesunate on cisplatin-resistant BCa was evaluated. Cisplatin-sensitive (parental) and cisplatin-resistant BCa cells, RT4, RT112, T24, and TCCSup, were treated with ART (1–100 µM). Cell growth, proliferation, and cell cycle phases were investigated, as were apoptosis, necrosis, ferroptosis, autophagy, metabolic activity, and protein expression. Exposure to ART induced a time- and dose-dependent significant inhibition of tumor cell growth and proliferation of parental and cisplatin-resistant BCa cells. This inhibition was accompanied by a G0/G1 phase arrest and modulation of cell cycle regulating proteins. ART induced apoptos is by enhancing DNA damage, especially in the resistant cells. ART did not induce ferroptosis, but led to a disturbance of mitochondrial respiration and ATP generation. This impairment correlated with autophagy accompanied by a decrease in LC3B-I and an increase in LC3B-II. Since ART significantly inhibits proliferative and metabolic aspects of cisplatin-sensitive and cisplatin-resistant BCa cells, it may hold potential in treating advanced and therapy-resistant BCa.
Leukotrienes constitute a group of bioactive lipids generated by the 5-lipoxygenase (5-LO) pathway. An increasing body of evidence supports an acute role for 5-LO products already during the earliest stages of pancreatic, prostate, and colorectal carcinogenesis. Several pieces of experimental data form the basis for this hypothesis and suggest a correlation between 5-LO expression and tumor cell viability. First, several independent studies documented an overexpression of 5-LO in primary tumor cells as well as in established cancer cell lines. Second, addition of 5-LO products to cultured tumor cells also led to increased cell proliferation and activation of anti-apoptotic signaling pathways. 5-LO antisense technology approaches demonstrated impaired tumor cell growth due to reduction of 5-LO expression. Lastly, pharmacological inhibition of 5-LO potently suppressed tumor cell growth by inducing cell cycle arrest and triggering cell death via the intrinsic apoptotic pathway. However, the documented strong cytotoxic off-target effects of 5-LO inhibitors, in combination with the relatively high concentrations of 5-LO products needed to achieve mitogenic effects in cell culture assays, raise concern over the assignment of the cause, and question the relationship between 5-LO products and tumorigenesis. Keywords: leukotriene, apoptosis, cell proliferation, mitogenic effects, cytotoxicity
Evasion of apoptosis, for example, by inhibitor of apoptosis (IAP) proteins, contributes to treatment resistance and poor outcome in acute myeloid leukemia (AML). Here we identify a novel synergistic interaction between the small-molecule second mitochondria-derived activator of caspases (Smac) mimetic BV6, which antagonizes X-linked IAP, cellular IAP (cIAP)1 and cIAP2, and the demethylating agents 5-azacytidine or 5-aza-2′-deoxycytidine (DAC) to induce cell death in AML cells, including apoptosis-resistant cells. Calculation of combination index (CI) confirms that this drug combination is highly synergistic (CI 0.02–0.4). In contrast, BV6 and DAC at equimolar concentrations do not cause synergistic toxicity against normal peripheral blood lymphocytes, pointing to some tumor cell selectivity. Molecular studies reveal that BV6 and DAC cooperate to trigger the activation of caspases, mitochondrial perturbations and DNA fragmentation, consistent with apoptotic cell death. However, the broad-range caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) fails to protect against BV6/DAC-induced cell death and even significantly increases the percentage of Annexin-V/propidium iodide double-positive cells. Importantly, BV6/DAC-induced cell death in the presence of zVAD.fmk is significantly reduced by pharmacological inhibition of key components of necroptosis signaling, that is, receptor-interacting protein (RIP) 1 using necrostatin-1 or mixed lineage kinase domain-like protein (MLKL) using necrosulfonamide. This indicates a switch from BV6/DAC-induced cell death from apoptosis to necroptosis upon caspase inhibition. Thus, BV6 cooperates with demethylating agents to induce cell death in AML cells and circumvents apoptosis resistance via a switch to necroptosis as an alternative mode of cell death. The identification of a novel synergism of BV6 and demethylating agents has important implications for the development of new treatment strategies for AML.
Smac mimetics antagonize IAP proteins, which are highly expressed in several cancers. Recent reports indicate that Smac mimetics trigger a broad cytokine response and synergize with immune modulators to induce cell death. Here, we identify a differential requirement of TRAIL or TNFα as mediators of IFNα/Smac mimetic-induced cell death depending on the cellular context. Subtoxic concentrations of Smac mimetics cooperate with IFNα to induce cell death in various solid tumor cell lines in a highly synergistic manner as determined by combination index. Mechanistic studies show that IFNα/BV6 cotreatment promotes the formation of a caspase-8-activating complex together with the adaptor protein FADD and RIP1. Assembly of this RIP1/FADD/caspase-8 complex represents a critical event, since RIP1 silencing inhibits IFNα/BV6-induced cell death. Strikingly, pharmacological inhibition of paracrine/autocrine TNFα signaling by the TNFα scavenger Enbrel rescues HT-29 colon carcinoma cells, but not A172 glioblastoma cells from IFNα/BV6-induced cell death. By comparison, A172 cells are significantly protected against IFNα/BV6 treatment by blockage of TRAIL signaling through genetic silencing of TRAIL or its cognate receptor TRAIL receptor 2 (DR5). Despite this differential requirement of TNFα and TRAIL signaling, mRNA and protein expression is increased by IFNα/BV6 cotreatment in both cell lines. Interestingly, A172 cells turn out to be resistant to exogenously added recombinant TNFα even in the presence of BV6, whereas they display a high sensitivity towards TRAIL/BV6. In contrast, BV6 efficiently sensitizes HT-29 cells to TNFα while TRAIL only had limited efficacy. This demonstrates that a differential sensitivity towards TRAIL or TNFα determines the dependency on either death receptor ligand for IFNα/Smac mimetic-induced cell death. Thus, by concomitant stimulation of both death receptor systems IFNα/Smac mimetic combination treatment is an effective strategy to induce cell death in TNFα- or TRAIL-responsive cancers.
Impact of Polo-like kinase 1 inhibitors on human adipose tissue-derived mesenchymal stem cells
(2016)
Polo-like kinase 1 (Plk1) has been established as one of the most promising targets for molecular anticancer intervention. In fact, various Plk1 inhibitors have been identified and characterized. While the data derived from the bench are prospective, the clinical outcomes are less encouraging by showing modest efficacy. One of the explanations for this discrepancy could be unintendedly targeting of non-malignant cells by Plk1 inhibitors. In this work, we have addressed the effect of Plk1 inhibition in adipose tissue-derived mesenchymal stem cells (ASCs). We show that both visceral and subcutaneous ASCs display monopolar spindles, reduced viability and strong apoptosis induction upon treatment with BI 2536 and BI 6727, the Plk1 kinase domain inhibitors, and with Poloxin, the regulatory Polo-box domain inhibitor. While Poloxin triggers quickly apoptosis, BI 2536 and BI 6727 result in mitotic arrest in ASCs. Importantly, survived ASCs exhibit DNA damage and a pronounced senescent phenotype. In addition, Plk1 inhibition impairs ASCs’ motility and homing ability. These results show that Plk1 inhibitors target slowly proliferating ASCs, an important population of anti-inflammation and immune modulation. The toxic effects on primary cells like ASCs could be partially responsible for the reported moderate antitumor activity in patients treated with Plk1 inhibitors.
Macrophages ingesting apoptotic cells attenuate inflammatory responses, such as reactive oxygen species (ROS) generation. In atherosclerosis, ongoing inflammation and accumulation of apoptotic/necrotic material are observed, suggesting defects of phagocytes in recognizing or responding to dying cells. Modified lipoproteins such as oxidized LDL (oxLDL) are known to promote inflammation and to interfere with apoptotic cell clearance. Here, we studied the impact of cells exposed to oxLDL on their ability to interfere with the oxidative burst in phagocytes. In contrast to apoptotic cells, cells dying in response to or in the presence of oxLDL failed to suppress ROS generation despite efficiently being taken up by phagocytes. In addition, apoptotic cells, but not oxLDL-treated cells, inhibited phosphorylation of extracellular signal-regulated kinase, which is important for NADPH oxidase activation. oxLDL treatment did not interfere with activation of the antiinflammatory transcriptional regulator peroxisome proliferator-activated receptor gamma by apoptotic cells. Moreover, cells exposed to oxLDL failed to suppress lipopolysaccharide- induced proinflammatory cytokine expression, whereas apoptotic cells attenuated these phagocyte responses. Thus, the presence of oxLDL during cell death impaired the ability of apoptotic cells to act antiinflammatory with regard to oxidative burst inhibition and cytokine expression in phagocytes.
Simple Summary:
Pharmacological activation of tumor suppressor p53 is a promising therapeutic strategy for a range of hematologic and solid cancers. Whether p53 activation augments or suppresses anti-tumor innate immunity is less understood. Here we show that treatment of differentiating human macrophages with a p53 activator idasanutlin suppresses their inflammatory responses to activators of toll-like receptors (TLR) -4 and -7/8. This is accompanied by reduced expression of TLR7, TLR8, as well as TLR4 co-receptor CD14. These data help evaluating the possibilities of combining p53-targeting and immunostimulatory anti-cancer therapies.
Abstract:
The transcription factor p53 has well-recognized roles in regulating cell cycle, DNA damage repair, cell death, and metabolism. It is an important tumor suppressor and pharmacological activation of p53 by interrupting its interaction with the ubiquitin E3 ligase mouse double minute 2 homolog (MDM2) is actively explored for anti-tumor therapies. In immune cells, p53 modulates inflammatory responses, but the impact of p53 on macrophages remains incompletely understood. In this study, we used the MDM2 antagonist idasanutlin (RG7388) to investigate the responses of primary human macrophages to pharmacological p53 activation. Idasanutlin induced a robust p53-dependent transcriptional signature in macrophages, including several pro-apoptotic genes. However, idasanutlin did not generally sensitize macrophages to apoptosis, except for an enhanced response to a Fas-stimulating antibody. In fully differentiated macrophages, idasanutlin did not affect pro-inflammatory gene expression induced by toll-like receptor 4 (TLR4), TLR3, and TLR7/8 agonists, but inhibited interleukin-4-induced macrophage polarization. However, when present during monocyte to macrophage differentiation, idasanutlin attenuated inflammatory responses towards activation of TLR4 and TLR7/8 by low doses of lipopolysaccharide or resiquimod (R848). This was accompanied by a reduced expression of CD14, TLR7, and TLR8 in macrophages differentiated in the presence of idasanutlin. Our data suggest anti-inflammatory effects of pharmacological p53 activation in differentiating human macrophages.
Efferocytosis is critical for tissue homeostasis, as its deregulation is associated with several autoimmune pathologies. While engulfing apoptotic cells, phagocytes activate transcription factors, such as peroxisome proliferator-activated receptors (PPAR) or liver X receptors (LXR) that orchestrate metabolic, phagocytic, and inflammatory responses towards the ingested material. Coordination of these transcription factors in efferocytotic human macrophages is not fully understood. In this study, we evaluated the transcriptional profile of macrophages following the uptake of apoptotic Jurkat T cells using RNA-seq analysis. Results indicated upregulation of PPAR and LXR pathways but downregulation of sterol regulatory element-binding proteins (SREBP) target genes. Pharmacological inhibition and RNA interference pointed to LXR and PPARδ as relevant transcriptional regulators, while PPARγ did not substantially contribute to gene regulation. Mechanistically, lysosomal digestion and lysosomal acid lipase (LIPA) were required for PPAR and LXR activation, while PPARδ activation also demanded an active lysosomal phospholipase A2 (PLA2G15). Pharmacological interference with LXR signaling attenuated ABCA1-dependent cholesterol efflux from efferocytotic macrophages, but suppression of inflammatory responses following efferocytosis occurred independently of LXR and PPARδ. These data provide mechanistic details on LXR and PPARδ activation in efferocytotic human macrophages.
Shikonin reduces growth of docetaxel-resistant prostate cancer cells mainly through necroptosis
(2021)
Simple Summary: Prostate carcinoma (PCa) is the most common tumor in men with an increasing age-associated risk. Several therapy strategies, one of which is docetaxel (DX) chemotherapy, have been established. However, due to the development of therapy resistance, in which chemotherapy no longer effectively combats the cancer, advanced, metastasized PCa with a poor prognosis may become manifested and therapy inevitably fails. Thus, new treatment options are urgently needed. Shikonin (SHI), from Traditional Chinese Medicine, has revealed promising antitumor activity in several tumor entities. In the current study, the impact of SHI on four therapy-sensitive and four respective DX-resistant PCa cell lines was determined. SHI induced growth inhibition mainly by necroptosis, a type of cell death, in all the tested therapy-sensitive, but more importantly, DX-resistant PCa cell lines. Corresponding molecular alterations contributing to growth inhibition after SHI exposure were found. SHI could, therefore, be a promising additive in treating advanced PCa.
Abstract: The prognosis for advanced prostate carcinoma (PCa) remains poor due to development of therapy resistance, and new treatment options are needed. Shikonin (SHI) from Traditional Chinese Medicine has induced antitumor effects in diverse tumor entities, but data related to PCa are scarce. Therefore, the parental (=sensitive) and docetaxel (DX)-resistant PCa cell lines, PC3, DU145, LNCaP, and 22Rv1 were exposed to SHI [0.1–1.5 μM], and tumor cell growth, proliferation, cell cycling, cell death (apoptosis, necrosis, and necroptosis), and metabolic activity were evaluated. Correspondingly, the expression of regulating proteins was assessed. Exposure to SHI time- and dose-dependently inhibited tumor cell growth and proliferation in parental and DX-resistant PCa cells, accompanied by cell cycle arrest in the G2/M or S phase and modulation of cell cycle regulating proteins. SHI induced apoptosis and more dominantly necroptosis in both parental and DX-resistant PCa cells. This was shown by enhanced pRIP1 and pRIP3 expression and returned growth if applying the necroptosis inhibitor necrostatin-1. No SHI-induced alteration in metabolic activity of the PCa cells was detected. The significant antitumor effects induced by SHI to parental and DX-resistant PCa cells make the addition of SHI to standard therapy a promising treatment strategy for patients with advanced PCa.
Simple Summary:
CDK9, in combination with Cyclin T1, is one of the major regulators of RNA Polymerase II mediated productive transcription of critical genes in any cell. The activity of CDK9 is significantly up-regulated in a wide variety of cancer entities, to aid in the overexpression of genes responsible for the regulation of functions, which are beneficial to the cancer cells, like proliferation, survival, cell cycle regulation, DNA damage repair and metastasis. Enhanced CDK9 activity, therefore, leads to poorer prognosis in many cancer types, offering the rationale to target it using small-molecule inhibitors. Several, increasingly specific inhibitors, have been developed, some of which are presently in clinical trials. Other approaches being tested involve combining inhibitors against CDK9 activity with those against CDK9’s upstream regulators like BRD4, SEC and HSP90; or downstream effectors like cMYC and MCL-1. The inhibition of CDK9’s activity holds the potential to be a highly effective anti-cancer therapeutic.
Abstract:
Cyclin Dependent Kinase 9 (CDK9) is one of the most important transcription regulatory members of the CDK family. In conjunction with its main cyclin partner—Cyclin T1, it forms the Positive Transcription Elongation Factor b (P-TEFb) whose primary function in eukaryotic cells is to mediate the positive transcription elongation of nascent mRNA strands, by phosphorylating the S2 residues of the YSPTSPS tandem repeats at the C-terminus domain (CTD) of RNA Polymerase II (RNAP II). To aid in this process, P-TEFb also simultaneously phosphorylates and inactivates a number of negative transcription regulators like 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) Sensitivity-Inducing Factor (DSIF) and Negative Elongation Factor (NELF). Significantly enhanced activity of CDK9 is observed in multiple cancer types, which is universally associated with significantly shortened Overall Survival (OS) of the patients. In these cancer types, CDK9 regulates a plethora of cellular functions including proliferation, survival, cell cycle regulation, DNA damage repair and metastasis. Due to the extremely critical role of CDK9 in cancer cells, inhibiting its functions has been the subject of intense research, resulting the development of multiple, increasingly specific small-molecule inhibitors, some of which are presently in clinical trials. The search for newer generation CDK9 inhibitors with higher specificity and lower potential toxicities and suitable combination therapies continues. In fact, the Phase I clinical trials of the latest, highly specific CDK9 inhibitor BAY1251152, against different solid tumors have shown good anti-tumor and on-target activities and pharmacokinetics, combined with manageable safety profile while the phase I and II clinical trials of another inhibitor AT-7519 have been undertaken or are undergoing. To enhance the effectiveness and target diversity and reduce potential drug-resistance, the future of CDK9 inhibition would likely involve combining CDK9 inhibitors with inhibitors like those against BRD4, SEC, MYC, MCL-1 and HSP90.