Ex vivo expansion of human hematopoietic stem cells by garcinol, a potent inhibitor of histone acetyltransferase

Background

Human cord blood (hCB) is the main source of hematopoietic stem and progenitor cells (HSCs/PCs) for transplantation. Efforts to overcome relative shortages of HSCs/PCs have led to technologies to expand HSCs/PCs ex vivo. However, methods suitable for clinical practice have yet to be fully established.

Methodology/Principal Findings

In this study, we screened biologically active natural products for activity to promote expansion of hCB HSCs/PCs ex vivo, and identified Garcinol, a plant-derived histone acetyltransferase (HAT) inhibitor, as a novel stimulator of hCB HSC/PC expansion. During a 7-day culture of CD34⁺CD38^– HSCs supplemented with stem cell factor and thrombopoietin, Garcinol increased numbers of CD34⁺CD38^– HSCs/PCs more than 4.5-fold and Isogarcinol, a derivative of Garcinol, 7.4-fold. Furthermore, during a 7-day culture of CD34⁺ HSCs/PCs, Garcinol expanded the number of SCID-repopulating cells (SRCs) 2.5-fold. We also demonstrated that the capacity of Garcinol and its derivatives to expand HSCs/PCs was closely correlated with their inhibitory effect on HAT. The Garcinol derivatives which expanded HSCs/PCs inhibited the HAT activity and acetylation of histones, while inactive derivatives did not.

Conclusions/Significance

Our findings identify Garcinol as the first natural product acting on HSCs/PCs and suggest the inhibition of HAT to be an alternative approach for manipulating HSCs/PCs.     

13,14-Dihydroxy groups are critical for the anti-cancer effects of garcinol

In the presence of K₂CO₃/Cs₂CO₃ (molar ratio 10:1), garcinol was subjected to methylation by reaction with iodomethane at room temperature to afford 13,14-dimethoxy garcinol. The methylated garcinol derivative was screened against oral cancer cell line SCC15 for cell proliferation and apoptosis. 13,14-Dimethoxy garcinol showed weaker inhibitory activity on SCC15 cell growth than garcinol, and had little effect on cell cycle and apoptosis of SCC15, whereas garcinol effectively induced cell cycle arrest and cell apoptosis. Meanwhile, the ELISA data showed that the inhibitory effect of garcinol on 5-Lox pathway was more potent than 13,14-dimethoxy garcinol (P < 0.05). All these results have confirmed the important role of 13,14-dihydroxy groups for anti-cancer effects of garcinol.     

Effects of garcinol on free radical generation and NO production in embryonic rat cortical neurons and astrocytes

Garcinol (camboginol) is a polyisoprenylated benzophenone derivative isolated from fruit rind of Garcinia indica. This study was to elucidate the anti-oxidative and neuroprotective properties of garcinol in rat cortical neuron cultures. First, garcinol protects DNA from Fenton reaction-induced breakage in a dose-dependent manner, with an IC(50) value of 0.32 microM. Garcinol also inhibits xanthine oxidase activity with an IC(50) value of 52 microM and exhibits competitive inhibition. To further ascertain the neuroprotective effects of garcinol in inflammatory-mediated neurotoxicity, we utilized primary neuron/astrocyte co-cultures treated with LPS or cytokine. Our data implicate that treatment with garcinol (5 microM) for 7 days promotes neuronal attachment and neurite extension. The formation of nitric oxide (NO) by LPS in rat astrocytes has been suggested to correlate with the neurodegenerative process. In identifying the effect of neuroprotection, we found that garcinol prevented NO accumulation in LPS-treated astrocytes. Garcinol significantly reduced the expression of LPS-induced inflammatory mediators, such as iNOS and COX-2. Consequently, our results suggest that the neuroprotective effects of garcinol are associated with anti-oxidation and inhibition of iNOS induction in astrocytic cells. Garcinol may exert a similar anti-inflammatory effect and may be neuroprotective against brain injury.     

Synthesis and biological activity of the probable biosynthetic precursors of 241-norbrassinolide

A series of 24¹-norbrassinolide biosynthetic precursors containing 3??-OH, 3-keto, ??²- or 2??,3??-epoxy functional groups less polar than 2??,3??-diol group in A-ring, and (22R,23R)-diol group in the side chain, was synthesized. Their biological activity as the proliferation regulators in MCF-7 human breast cancer and LNCaP human prostate adenocarcinoma cells was studied. It was shown that the majority of the derivatives obtained effectively suppress cell proliferation. The dependence of proliferation on the concentration of the studied compounds was found in LNCaP human prostate adenocarcinoma cells (IC₅₀ = 13?C28 ??M at 72 h of incubation in a medium containing 10% FBS, and suppression of DNA biosynthesis). Some compounds stimulated apoptosis (23?C33%), blocked the cell cycle in S- and G2/M-phases, and induced a partial detachment of cells during a prolonged incubation.     

The Selective Target of Capsaicin on FASN Expression and De Novo Fatty Acid Synthesis Mediated through ROS Generation Triggers Apoptosis in HepG2 Cells

The inhibition of the mammalian de novo synthesis of long-chain saturated fatty acids (LCFAs) by blocking the fatty acid synthase (FASN) enzyme activity in tumor cells that overexpress FASN can promote apoptosis, without apparent cytotoxic to non-tumor cells. The present study aimed to focus on the potent inhibitory effect of capsaicin on the fatty acid synthesis pathway inducing apoptosis of capsaicin in HepG2 cells. The use of capsaicin as a source for a new FASN inhibitor will provide new insight into its possible application as a selective anti-cancer therapy. The present findings showed that capsaicin promoted apoptosis as well as cell cycle arrest in the G0/G1 phase. The onset of apoptosis was correlated with a dissipation of mitochondrial membrane potential (ΔΨm). Apoptotic induction by capsaicin was mediated by inhibition of FASN protein expression which was accompanied by decreasing its activity on the de novo fatty acid synthesis. The expression of FASN was higher in HepG2 cells than in normal hepatocytes that were resistant to undergoing apoptosis following capsaicin administration. Moreover, the inhibitory effect of capsaicin on FASN expression and activity was found to be mediated by an increase of intracellular reactive oxygen species (ROS) generation. Treatment of HepG2 cells with capsaicin failed to alter ACC and ACLY protein expression, suggesting ACC and ACLY might not be the specific targets of capsaicin to induce apoptosis. An accumulation of malonyl-CoA level following FASN inhibition represented a major cause of mitochondrial-dependent apoptotic induction instead of deprivation of fatty acid per se. Here, we also obtained similar results with C75 that exhibited apoptosis induction by reducing the levels of fatty acid without any change in the abundance of FASN expression along with increasing ROS production. Collectively, our results provide novel evidence that capsaicin exhibits a potent anti-cancer property by targeting FASN protein in HepG2 cells.     

Myricetin exhibit selective anti-lymphoma activity by targeting BTK and is effective via oral administration in vivo

BackgroundMyricetin (MYR) is a polyhydroxy flavone originally isolated from Myrica rubra, and is widely distributed in a variety of medicinal plants and delicious food. MYR has been proven to have inhibitory effects against various types of cancer. However, the exact role of MYR in lymphoma development is still unclear.MethodsIn vitro, the MTT assay was performed to evaluate the activity of human diffuse large B lymphoma cell TMD-8 and other tumor cells. Homogeneous time-resolved fluorescence (HTRF) and molecular docking were used to detect the target of MYR inhibiting TMD-8 cells. In addition, flow cytometry, Annexin V-FITC/PI assays, Hoechst 33258, and mondansylcadaverine (MDC) fluorescent standing were used to detect the cell cycle, apoptosis, and autophagy, respectively. Moreover, Western blot analysis was conducted to analyze related signaling pathways. In TMD-8 cell xenotransplanted mice, immunohistochemistry, histopathology, and blood biochemical tests were used to evaluate the effectiveness and safety of oral administration of MYR.ResultsHere, we found that MYR is more sensitive to TMD-8 cells than other tumor cells by targeting bruton tyrosine kinase (BTK). BTK is an attractive target for the treatment of B-cell malignancies. The HTRF assay showed that MYR inhibited BTK kinase with an IC₅₀ of 1.82 μM. Furthermore, the HTRF assay and Western blot analysis demonstrated that MYR could bind to key residues (Ala478, Leu408, Thr474) in the BTK active pocket, inhibit the autophosphorylation on tyrosine 223, and block BTK/ERK and BTK/AKT signal transduction cascades (including downstream substrates GSK-3β, IKK, STAT3, and NF-κb). The results of cell cycle, apoptosis, and autophagy showed that MYR could induce G1/G0 cycle arrest by regulating cyclinB1/D1 expression, induce apoptosis by increasing the Bax/Bcl-2 ratio, and trigger autophagy by inhibiting mTOR activation. In vivo, oral administration of MYR significantly inhibited the growth of TMD-8 xenograft tumora without toxic side effects. Furthermore, Ki67 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis showed that MYR could inhibit proliferation and induce apoptosis of tissue lymphoma cells.ConclusionTaken together, MYR is an oral available natural BTK inhibitor that effectively inhibits the growth of lymphoma TMD-8 cells both in vitro and in vivo. In addition, our findings support that the use of MYR is a novel and promising therapeutic strategy for the treatment of lymphoma.     

Discovery of 18β-glycyrrhetinic acid conjugated aminobenzothiazole derivatives as Hsp90-Cdc37 interaction disruptors that inhibit cell migration and reverse drug resistance

A series of 18β-glycyrrhetinic acid (GA) conjugated aminobenzothiazole derivatives were designed, synthesized and evaluated for disruption activity of Hsp90-Cdc37 as well as the effects of in vitro cell migration. These compounds exhibited relatively good disruption activity against Hsp90-Cdc37 with IC₅₀ values in low micromolar range. A docking study of the most active compound 11g revealed key interactions between 11g and Hsp90-Cdc37 complex in which the benzothiazole moiety and the amine chain group were important for improving activity. It is noteworthy that further antitumor activity screening revealed that some compounds exhibited better inhibitory activity than the commercial anticancer drug 5-FU and showed potent suppression activity against drug-resistant cancer cells. In particular, compound 11?g appeared to be the most potent compound against the A549 cell line, at least partly, by inhibition of the activity of Hsp90 and apoptosis induction. The treatment of A549 cells with compound 11g resulted in inhibition of in vitro cell migration through wound healing assay and S phase of cell cycle arrested. In addition, 11g-induced apoptosis was significantly facilitated in A549 cells. Thus, we conclude that GA aminobenzothiazole derivatives may be the potential Hsp90-Cdc37 disruptors with the ability to suppress cells migration and reversed drug-resistant.     

Inhibition of non-muscle myosin II leads to G0/G1 arrest of Wharton's jelly-derived mesenchymal stromal cells

Background aimsMesenchymal stromal cells (MSCs) have remarkable clinical potential for cell-based therapy. Wharton's jelly-derived mesenchymal stromal cells (WJ-MSCs) from umbilical cord share unique properties with both embryonic and adult stem cells. MSCs are found at low frequency in vivo, and their successful therapeutic application depends on rapid and efficient large-scale expansion in vitro. Non-muscle myosin II (NMII) has pivotal roles in different cellular activities, such as cell division, migration and differentiation. We performed this study to understand the role of NMII in proliferation and cell cycle progression in WJ-MSCs.MethodsWJ-MSCs were cultured in the presence of blebbistatin, and cell cycle analysis was performed using flow cytometry, proliferation kinetics, senescence assay and gene expression profile using polymerase chain reaction array.ResultsWhen cultured in the presence of blebbistatin, an inhibitor of NMII adenosine triphosphatase activity, WJ-MSCs exhibited dose-dependent reduction in proliferative potential along with increase in cell size and induction of early senescence. Inhibition of NMII activity also affected cell cycle progression in WJ-MSCs and led to an increase in the percentage of cells in G₀/G₁ phase with a corresponding reduction in the percentage of cells in G₂/M phase. Blebbistatin-induced G₀/G₁ arrest of WJ-MSCs was further associated with up-regulation of cell cycle inhibitory genes CDKN1A, CDKN2A and CDKN2B and down-regulation of numerous genes related to progression through S and M phases of the cell cycle.ConclusionsOur study demonstrates that inhibition of NMII activity in WJ-MSCs leads to G₀/G₁ arrest and alteration in the expression levels of certain key cell cycle-related genes.     

Coupling of the Cell Cycle and Apoptotic Machineries in Developing T Cells

Proliferation and apoptosis are diametrically opposite processes. Expression of certain genes like c-Myc, however, can induce both, pointing to a possible linkage between them. Developing CD4⁺CD8⁺ thymocytes are intrinsically sensitive to apoptosis, but the molecular basis is not known. We have found that these noncycling cells surprisingly express many cell cycle proteins. We generated transgenic mice expressing a CDK2 kinase-dead (CDK2-DN) protein in the T cell compartment. Analysis of these mice showed that the CDK2-DN protein acts as a dominant negative mutant in mature T cells as expected, but surprisingly, it acts as a dominant active protein in CD4⁺CD8⁺ thymocytes. The levels of CDK2 kinase activity, cyclin E, cyclin A, and other cell cycle proteins in transgenic CD4⁺CD8⁺ thymocytes are increased. Concurrently, caspase levels are elevated, and apoptosis is significantly enhanced in vitro and in vivo. E2F-1, the unique E2F member capable of inducing apoptosis when overexpressed, is specifically up-regulated in transgenic CD4⁺CD8⁺ thymocytes but not in other T cell populations. These results demonstrate that the cell cycle and apoptotic machineries are normally linked, and expression of cell cycle proteins in developing T cells contributes to their inherent 1sensitivity to apoptosis.     

Shogaols at proapoptotic concentrations induce G<Subscript>2</Subscript>/M arrest and aberrant mitotic cell death associated with tubulin aggregation

Shogaols have been previously reported to induce cancer cell death via multiple mechanisms, among which one analog 6-shogaol has been reported to cause microtubule damage through specific reaction with sulfhydryl groups in tubulin. In this study, a series of shogaols with different side chain lengths (4-, 6-, 8- and 10-shogaol) was synthesized and evaluated for antiproliferative activity in HCT 116 colon carcinoma and SH-SY5Y neuroblastoma cells. 4- and 6-shogaol were identified as lead compounds possessing the strongest antiproliferative activity. In the soft agar assay, the lead shogaols displayed dose-dependent inhibition on cancer cell colony formation under anchorage-independent conditions. Using HCT 116 as the selected cancer cell line, the molecular events linking shogaols-induced G₂/M cell cycle arrest to apoptosis characterized by caspase 3 and PARP cleavage were investigated. At sublethal concentrations, the halt at G₂/M phase was alleviated along time and cells survived. Conversely, proapoptotic concentrations of 4- and 6-shogaol induced irreversible G₂/M arrest that was at least in part associated with down-regulation of cell cycle checkpoint proteins cdk1, cyclin B and cdc25C, as well as spindle assembly checkpoint proteins mad2, cdc20 and survivin. A dose- and time-dependent accumulation of insoluble tubulin in the insoluble fractions of cell lysates provided evidence that G₂ checkpoint failure led to disruption of microtubule turnover. In summary, our results conclude that shogaols cause apoptosis by inducing aberrant mitosis at least through the attenuation of cell cycle and spindle assembly checkpoint proteins.     

Mechanism of apoptosis induced by a newly synthesized derivative of macrosphelides with a thiazole side chain

The apoptosis-inducing ability of hybrid compounds composed of macrosphelide and thiazole-containing side chain of epothilones was investigated. Among the tested series of hybrid compounds the one containing thiazole side chain at C15 (MSt-2) showed the maximum potency to induce apoptosis, while another containing thiazole side chain at C3 (MSt-6) was less potent. MSt-2 was found to induce apoptosis in human lymphoma (U937) cells in a dose- and time-dependent manner as confirmed by DNA fragmentation analysis. MSt-2 treated cells showed rapid reactive oxygen species (ROS) formation and c-Jun N-terminal kinase (JNK) activation. Furthermore, significant activation of extrinsic pathway as evident by Fas expression and caspase-8 activation was also observed. MSt-2-mediated decreased expression of Bid is an important event for cross talk between intrinsic and extrinsic signaling. N-acetyl-l-cysteine pre-treatment rescued cells from MSt-2-induced ROS formation, mitochondrial membrane potential (Δψ_m) loss, Fas expression, caspase-8 and -3 activation and DNA fragmentation. Moreover, antioxidant enzymes catalase and/or superoxide dismutase conjugated with polyethylene glycol also inhibit MSt-2-induced ROS formation, apoptosis and Δψ_m loss suggesting thereby pro-oxidant effects of MSt-2. Furthermore, JNK and pan-caspase inhibitors also protect cells from MSt-2-induced apoptosis. In addition to this, MSt-2 was found to be more potent in human colon carcinoma (HCT116) and human gastric cancer (AGS) cells while it has no effect on human normal dermal fibroblast. The important structure-activity relationship observed in the current study which makes MSt-2 more potent than MSt-6 is the position of thiazole side chain and stereochemistry of position 3 in chemical structure. In short the results of our study demonstrate that MSt-2-induced rapid ROS generation and mitochondrial dysfunction in cells trigger events responsible for mitochondria-dependent apoptosis pathway.     

Role of potassium channels in chlorogenic acid-induced apoptotic volume decrease and cell cycle arrest in Candida albicans

BackgroundChlorogenic acid (CRA) is an abundant phenolic compound in the human diet. CRA has a potent antifungal effect, inducing cell death in Candida albicans. However, there are no further studies to investigate the antifungal mechanism of CRA, associated with ion channels.MethodsTo evaluate the inhibitory effects on CRA-induced cell death, C. albicans cells were pretreated with potassium and chloride channel blockers, separately. Flow cytometry was carried out to detect several hallmarks of apoptosis, such as cell cycle arrest, caspase activation, and DNA fragmentation, after staining of the cells with SYTOX green, FITC-VAD-FMK, and TUNEL.ResultsCRA caused excessive potassium efflux, and an apoptotic volume decrease (AVD) was observed. This change, in turn, induced cytosolic calcium uptake and cell cycle arrest in C. albicans. Moreover, CRA induced caspase activation and DNA fragmentation, which are considered apoptotic markers. In contrast, the potassium efflux and proapoptotic changes were inhibited when potassium channels were blocked, whereas there was no inhibitory effect when chloride channels were blocked.ConclusionsCRA induces potassium efflux, leading to AVD and G2/M cell cycle arrest in C. albicans. Therefore, potassium efflux via potassium channels regulates the CRA-induced apoptosis, stimulating several apoptotic processes.General significanceThis study improves the understanding of the antifungal mechanism of CRA and its association with ion homeostasis, thereby pointing to a role of potassium channels in CRA-induced apoptosis.     

Discovery of indoline derivatives that inhibit esophageal squamous cell carcinoma growth by Noxa mediated apoptosis

A series of novel indoline derivatives were synthesized and evaluated for antiproliferative activity against four selected cancer cell lines (Hela, A549, HepG2 and KYSE30). Among them, compound 20 displayed the potent inhibition activity against esophageal cancer cells (Kyse30, Kyse450, Kyse510 and EC109). Cellular mechanism studies in esophageal squamous cell carcinoma (ESCC) cells elucidated compound 20 inhibited cell growths in vitro and in vivo, reduced colony formation, arrested cell cycle at M phase, and induced Noxa-dependent apoptosis in ESCC. Importantly, compound 20 was identified as a novel Noxa mediated apoptosis inducer. These results suggested that compound 20 might be a promising anticancer agent with potential for development of further clinical applications.     

Mechanism-based inhibitors of cytokinin oxidase/dehydrogenase attack FAD cofactor

Cytokinin oxidases/dehydrogenases (CKOs) mediate catabolic regulation of cytokinin levels in plants. Several substrate analogs containing an unsaturated side chain were studied for their possible inhibitory effect on maize CKO (ZmCKO1) by use of various bioanalytical methods. Two allenic derivatives, N⁶-(buta-2,3-dienyl)adenine (HA-8) and N⁶-(penta-2,3-dienyl)adenine (HA-1), were identified as strong mechanism-based inhibitors of the enzyme. Despite exhaustive dialysis, the enzyme remained inhibited. Conversely, substrate analogs with a triple bond in the side chain were much weaker inactivators. The crystal structures of recombinant ZmCKO1 complexed with HA-1 or HA-8 were solved to 1.95 Å resolution. Together with Raman spectra of the inactivated enzyme, it was revealed that reactive imine intermediates generated by oxidation of the allenic inhibitors covalently bind to the flavin adenine dinucleotide (FAD) cofactor. The binding occurs at the C4a atom of the isoalloxazine ring of FAD, the planarity of which is consequently disrupted. All the compounds under study were also analyzed for binding to the Arabidopsis cytokinin receptors AHK3 and AHK4 in a bacterial receptor assay and for cytokinin activity in the Amaranthus bioassay. HA-1 and HA-8 were found to be good receptor ligands with a significant cytokinin activity. Nevertheless, due to their ability to inactivate CKO in the desired time intervals or developmental stages, they both represent attractive compounds for physiological studies, as the inhibition mechanism of HA-1 and HA-8 is mainly FAD dependent.     

Effect of artemisinin on proliferation and apoptosis-related protein expression in vivo and in vitro

Artemisinin is the first-line drugs for the treatment of malaria. In recent years, a large number of reports showed that artemisinin exhibit anti-tumor activity. In this study, we used C6 glioma cells and rat C6 brain-glioma model to study anti-tumor activity of artemisinin in vivo and in vitro. We found that artemisinin inhibited the proliferation in C6 cells and induced cell cycle arrest and a caspase-3-dependent cell apoptosis. It also inhibited the growth of C6 brain-glioma in vivo and enhanced living state of rat brain-glioma model. These results suggested that artemisinin had significant anti-tumor activities on C6 cells both in vitro and in vivo. Artemisinin might be exploited as a promising clinical anti-cancer drug in future.     

Effects of garcinol and its derivatives on intestinal cell growth: Inhibitory effects and autoxidation-dependent growth-stimulatory effects

Garcinol, a polyisoprenylated benzophenone, from the Garcinia indica fruit rind, has been suggested to be an anti-inflammatory and anti-cancer agent. To explore the possible use of this redox-sensitive compound as a colon cancer preventive agent, we investigated the effects of garcinol and its oxidative derivatives, cambogin, garcim-1, and garcim-2, on the growth of HT-29 and HCT-116 colon cancer cells, as well as IEC-6 and INT-407 normal immortalized intestinal cells. Garcinol and its derivatives showed potent growth-inhibitory effects on all intestinal cells, showing IC50 of 3.2-21.4 microM after a 3-day treatment. Garcim-1 exhibited the strongest effect with IC50 of 3.2-5.9 microM. Garcinol was more effective in inhibiting growth of cancer cells than that of normal immortalized cells. Flow-cytometric analysis showed increased sub-G1 cells by treatment with garcinol and cambogin. Induction of apoptosis by garcinol and cambogin (2-10 microM) was also observed based on caspase-3 activation and enhanced annexin V staining. The inhibitory effect of garcinol on cell growth was much more pronounced in the absence of fetal bovine serum (FBS), decreasing IC50 to 1.5 from 11.8 microM in 72-h incubations and to 3 from 38 microM in 24-h incubations, possibly due to the binding of garcinol to FBS, which markedly reduced cellular levels of garcinol. Under these conditions, redox reactions seem not to be involved in the inhibition. In contrast to the inhibitory effect, low concentrations (<1 microM) of garcinol and cambogin stimulated the growth of both normal and cancer cells by 10-100%, and the activity seemed to be mediated by reactive oxygen species. In the presence of superoxide dismutase/catalase or N-acetyl cysteine, low concentrations of garcinol (<1 microM) decreased cell growth. Garcinol (0.5-1 microM) also increased the phosphorylation of extracellular signal-related kinase 1/2 and AKT and the level of survivin, and the effects were abolished in the presence of superoxide dismutase/catalase. Our results indicate that garcinol and its derivatives can inhibit intestinal cell growth, but low concentrations of garcinol can stimulate cell growth. It remains to be determined whether the currently observed stimulatory and inhibitory effects of garcinol on colon cell growth occur in vivo.     

New thiazol-hydrazono-coumarin hybrids targeting human cervical cancer cells: Synthesis,CDK2 inhibition,QSAR and molecular docking studies

Motivated by the potential anticancer activity of both coumarin and 2-aminothiazole nuclei, a new set of thiazol-2-yl hydrazono-chromen-2-one analogs were efficiently synthesized aiming to obtain novel hybrids with potential cytotoxic activity. MTT assay investigated the significant potency of all the target compounds against the human cervical cancer cell lines (HeLa cells). Cell cycle analysis showed that the representative compound 8a led to cell cycle cessation at G0/G1 phase indicating that CDK2/E1complex could be the plausible biological target for these newly synthesized compounds. Thus, the most active compounds (7c and 8a-c) were tested for their CDK2 inhibitory activity. The biological results revealed their significant CDK2 inhibitory activity with IC₅₀ range of 0.022–1.629 nM. Moreover, RT-PCR gene expression assay showed that compound 8a increased the levels of the nuclear CDK2 regulators P21 and P27 by 2.30 and 5.7 folds, respectively. ELISA tequnique showed also that compound 8a led to remarkable activation of caspases-9 and -3 inducing cell apoptosis. QSAR study showed that the charge distribution and molecular hydrophobicity are the structural features affecting cytotoxic activity in this series. Molecular docking study for the most potent cytotoxic compounds (7c and 8a-c) rationalized their superior CDK2 inhibitory activity through their hydrogen bonding and hydrophobic interactions with the key amino acids in the CDK2 binding site. Pharmacokinetic properties prediction of the most potent compounds showed that the newly synthesized compounds are not only with promising antitumor activity but also possess promising pharmacokinetic properties.     

Induction of apoptosis by magnolol via the mitochondrial pathway and cell cycle arrest in renal carcinoma cells

Magnolol (Mag), an effective natural compound isolated from the stem bark of Magnolia officinalis, was found to have the potential for antitumor activity by inducing apoptosis in tumor cells. However, the effect of Mag on renal carcinoma cells and its molecular mechanism are unexplored. Our study provided evidence that Mag induced apoptosis in 786-O and OS-RC-2?cell lines via the mitochondrial pathway and cell cycle arrest. In this work, we found that Mag induced morphological changes and inhibited the proliferation of 786-O and OS-RC-2?cells in a dose- and time-dependent manner but exerted no notable inhibitory effects on normal human renal proximal tubular (HK-2) cells. Treatment with Mag suppressed the migration and invasion ability of renal carcinoma cells. Moreover, Mag caused the openness of mPTP, the accumulation of intracellular ROS and decreased △Ψm, leading to mitochondrial dysfunction. However, pretreatment with the antioxidant N-acetyl cysteine (NAC) reversed the apoptosis induced by Mag and decreased the generation of ROS. In addition, the increased proportion of the G1/G0 phase indicated that Mag caused cell cycle arrest. Further analyses suggested that magnolol-induced apoptosis was related to the abnormal expression of p53, Bax, Bcl-2, cytochrome c and caspase activation. Together, the results above revealed that Mag had antitumor effects in renal carcinoma cells via ROS production as well as cell cycle arrest and the apoptotic mitochondrial pathway was suppressed in part by NAC.     

Formic Acid and Acetic Acid Induce a Programmed Cell Death in Pathogenic Candida Species

Cutaneous fungal infections are common and widespread. Antifungal agents used for the treatment of these infections often have undesirable side effects. Furthermore, increased resistance of the microorganisms to the antifungal drugs becomes the growing problem. Accordingly, the search for natural antifungal compounds continues to receive attention. Apoptosis is highly regulated programmed cell death. During yeast cell apoptosis, amino acids and peptides are released and can stimulate regeneration of human epithelium cells. Thus, detection of chemical compounds inducing apoptosis in yeast and nontoxic for humans is of great medical relevance. The aim of this study was to detect chemical compound inducing apoptosis in pathogenic Candida species with the lowest toxicity to the mammalian cells. Five chemical compounds—acetic acid, sodium bicarbonate, potassium carbonate, lithium acetate, and formic acid—were tested for evaluation of antifungal activity on C. albicans, C. guilliermondii, and C. lusitaniae. The results showed that acetic acid and formic acid at the lowest concentrations induced yeast cells death. Apoptosis analysis revealed that cells death was accompanied by activation of caspase. Minimal inhibitory concentrations of potassium carbonate and sodium bicarbonate induced Candida cells necrosis. Toxicity test with mammalian cell cultures showed that formic acid has the lowest effect on the growth of Jurkat and NIH 3T3 cells. In conclusion, our results show that a low concentration of formic acid induces apoptosis-like programmed cell death in the Candida yeast and has a minimal effect on the survivability of mammalian cells, suggesting potential applications in the treatment of these infections.     

Granzyme M targets topoisomerase II alpha to trigger cell cycle arrest and caspase-dependent apoptosis

Cytotoxic lymphocyte protease granzyme M (GrM) is a potent inducer of tumor cell death. The apoptotic phenotype and mechanism by which it induces cell death, however, remain poorly understood and controversial. Here, we show that GrM-induced cell death was largely caspase-dependent with various hallmarks of classical apoptosis, coinciding with caspase-independent G2/M cell cycle arrest. Using positional proteomics in human tumor cells, we identified the nuclear enzyme topoisomerase II alpha (topoIIα) as a physiological substrate of GrM. Cleavage of topoIIα by GrM at Leu¹²⁸⁰ separated topoIIα functional domains from the nuclear localization signals, leading to nuclear exit of topoIIα catalytic activity, thereby rendering it nonfunctional. Similar to the apoptotic phenotype of GrM, topoIIα depletion in tumor cells led to cell cycle arrest in G2/M, mitochondrial perturbations, caspase activation, and apoptosis. We conclude that cytotoxic lymphocyte protease GrM targets topoIIα to trigger cell cycle arrest and caspase-dependent apoptosis.