TPGS nanoparticles co-loaded with ABT-737 and R848 for breast cancer therapy.

The development of new effective drugs to treat breast cancer remains a huge challenge. ABT-737 can inhibit Bcl-2 proteins to promote apoptosis. Resiquimod (R848) is a TLR7/8 agonist that is effective in modulating the immunosuppressive microenvironment. In this study, a codelivery system (TPGS/ABT+R848 NPs) based on D-α-tocopheryl poly (ethylene glycol) 1000 succinate as a potential drug delivery vector to codelivery ABT-737 and R848 was investigated. The size of TPGS/ABT+R848 NPs was 102.5 nm, the drug loading of ABT-737 and R848 was 30.6 % and 12.5 %, and the entrapment efficiency was 84.2 % and 23.7 %, respectively. The nanoparticles showed no significant change in particle size over 14 days. R848 and ABT-737 were released in co-loaded nanoparticles in sequential order. In vitro anti-tumor experiments, the IC50 value of TPGS/ABT+R848 NPs was 0.30 µg·mL-1, 34 times lower than that of free ABT-737. Animal experiments also verified that TPGS/ABT+R848 NPs could enhance the anti-tumor activity, and the tumor weight inhibition rate was 75.3 %. This study demonstrated that TPGS NPs loaded with ABT-737 and R848 have superior combination tumor therapeutic effects, and the co-loaded preparation is conducive to anti-tumor efficacy. The TPGS/ABT+R848 NPs could be a promising platform against breast cancer.

ABT­737 increases cisplatin sensitivity through the ROS­ASK1­JNK MAPK signaling axis in human ovarian cancer cisplatin­resistant A2780/DDP cells.

Ovarian cancer is a gynecological malignant tumor with the highest mortality rate, and chemotherapy resistance seriously affects patient therapeutic outcomes. It has been shown that the high expression of anti­apoptotic proteins Bcl­2 and Bcl­xL is closely related to ovarian cancer chemotherapy resistance. Therefore, reducing Bcl­2 and Bcl­xL expression levels may be essential for reversing drug resistance in ovarian cancer. ABT­737 is a BH3­only protein mimetic, which can effectively inhibit the expression of the anti­apoptotic proteins Bcl­xL and Bcl­2. Although it has been shown that ABT­737 can increase the sensitivity of ovarian cancer cells to cisplatin, the specific molecular mechanism remains unclear and requires further investigation. In the present study, the results revealed that ABT­737 can significantly increase the activation levels of JNK and ASK1 induced by cisplatin in A2780/DDP cells, which are cisplatin­resistant ovarian cancer cells. Inhibition of the JNK and ASK1 pathway could significantly reduce cisplatin cytotoxicity increased by ABT­737 in A2780/DDP cells, while inhibiting the ASK1 pathway could reduce JNK activation. In addition, it was further determined that ABT­737 could increase reactive oxygen species (ROS) levels in A2780/DDP cells induced by cisplatin. Furthermore, the inhibition of ROS could significantly reduce JNK and ASK1 activation and ABT­737­mediated increased cisplatin cytotoxicity in A2780/DDP cells. Overall, the current data identified that activation of the ROS­ASK1­JNK signaling axis plays an essential role in the ability of ABT­737 to increase cisplatin sensitivity in A2780/DDP cells. Therefore, upregulation the ROS­ASK1­JNK signaling axis is a potentially novel molecular mechanism by which ABT­737 can enhance cisplatin sensitivity of ovarian cancer cells. In addition, the present research can also provide new therapeutic strategies and new therapeutic targets for patients with cisplatin­resistant ovarian cancer with high Bcl­2/Bcl­xL expression patterns.

[Melatonin Enhances the Effect of ABT-737 in Acute Monocytic Leukemia THP-1 Cells].

Melatonin (N-acetyl-5-methoxytryptamine, MEL) is a hormone synthesized by the pineal gland. Due to its oncostatic effect, it can be considered as an antitumor agent and used for combination therapy. ABT-737, a Bcl-2 inhibitor, promotes cell death after treatment with agents that induce pro-apoptotic signals. In the present study, the combined effect of MEL and ABT-737 on changes in proliferative and mitotic activity, mitochondrial membrane potential, intracellular production of reactive oxygen species (ROS), and cytosolic Ca^(2+) was studied. Moreover, changes in the expression of anti- and pro-apoptotic proteins (Bcl-2 and Bax), autophagy markers (LC3A/B (I, II)), endoplasmic reticulum stress markers (chaperones BIP and PDI, CHOP) were studied under these conditions. The effect of MEL together with ABT-737 led to an increase in the level of cytosolic Ca^(2+), intracellular production of ROS and a decrease in the membrane potential of mitochondria. The content of Bcl-2 increased, while the level of Bax decreased. Activation of CHOP stimulated autophagy and led to a decrease in the synthesis of chaperones BIP and PDI. It is assumed that melatonin can enhance the effect of other chemotherapeutic agents and can be used in the treatment of tumors.

Light-inducible nanodrug-mediated photodynamic and anti-apoptotic synergy for enhanced immunotherapy in triple-negative breast cancer.

Triple negative breast cancer (TNBC) exhibits limited responsiveness to immunotherapy owing to its immunosuppressive tumor microenvironment (TME). Here, a reactive oxygen species (ROS)-labile nanodrug encapsulating the photosensitizer Ce6 and Bcl-2 inhibitor ABT-737 was developed to provoke a robust immune response via the synergistic effect of photodynamic therapy (PDT) and the reversal of apoptosis resistance. Upon exposure to first-wave near-infrared laser irradiation, the generated ROS triggers PEG cleavage, facilitating the accumulation of the nanodrug at tumor region and endocytosis by tumor cells. Further irradiation leads to the substantial generation of cytotoxic ROS, initiating an immunogenic cell death (ICD) cascade, which prompts the maturation of dendritic cells (DCs) as well as the infiltration of T cells into the tumor site. Meanwhile, Bcl-2 inhibition counteracts apoptosis resistance, thereby amplifying PDT-induced ICD and bolstering antitumor immunity. As a result, the ROS-sensitive nanodrug demonstrates a potent inhibitory effect on tumor growth.

Catechol-O-Methyltransferase inhibition and alcohol use disorder: Evaluating the efficacy of tolcapone in ethanol-dependent rats.

Alcohol Use Disorder (AUD) is a significant public health issue in the United States. It affects millions of individuals and their families and contributes to substantial societal and economic burdens. Despite the availability of some pharmacological treatments, there is still a pressing need to develop more effective therapeutic strategies to address the diverse range of symptoms and challenges associated with AUD. Catechol-O-methyltransferase (COMT) inhibition recently emerged as a promising new approach to treating AUD due to its potential to improve cognitive effects commonly associated with AUD. Tolcapone, an FDA-approved COMT inhibitor, has shown some promise for treating AUD; however, its ability to decrease drinking in ethanol-dependent rats has not been well-established. In this study, we evaluated the effects of tolcapone on operant, oral ethanol self-administration in non-dependent and dependent rats, and in rats that self-administered oral saccharin. To induce dependence, rats underwent the chronic intermittent exposure to vapor model, and their drinking levels were assessed during acute withdrawal from ethanol. Our results demonstrated that tolcapone attenuated responding for ethanol in dependent rats only, without affecting self-administration in non-dependent rats or rats self-administering saccharin. Moreover, we found that tolcapone was differentially effective in different estrous phases in female rats. These findings suggest that COMT inhibition, specifically using tolcapone, may be a valuable pharmacotherapy for treating AUD, particularly in individuals who are physically dependent on alcohol. Further research is needed to elucidate the precise mechanisms underlying the observed effects and to assess the potential of COMT inhibitors in a broader population of individuals with AUD.

Melatonin Can Enhance the Effect of Drugs Used in the Treatment of Leukemia.

Melatonin (N-acetyl-5-methoxytryptamine, MEL), secreted by the pineal gland, plays an important role in regulation of various functions in the human body. There is evidence that MEL exhibits antitumor effect in various types of cancer. We studied the combined effect of MEL and drugs from different pharmacological groups, such as cytarabine (CYT) and navitoclax (ABT-737), on the state of the pool of acute myeloid leukemia (AML) tumor cell using the MV4-11 cell line as model. The combined action of MEL with CYT or ABT-737 contributed to the decrease in proliferative activity of leukemic cells, decrease in the membrane potential of mitochondria, and increase in the production of reactive oxygen species (ROS) and cytosolic Ca2+. We have shown that introduction of MEL together with CYT or ABT-737 increases expression of the C/EBP homologous protein (CHOP) and the autophagy marker LC3A/B and decreases expression of the protein disulfide isomerase (PDI) and binding immunoglobulin protein (BIP), and, therefore, could modulate endoplasmic reticulum (ER) stress and initiate autophagy. The findings support an early suggestion that MEL is able to provide benefits for cancer treatment and be considered as an adjunct to the drugs used in cancer therapy.

IFN-γ enhances CLL cell resistance to ABT-199 by regulating MCL-1 and BCL-2 expression via the JAK-STAT3 signaling pathway.

Although clinical outcomes of CLL have improved with the use of BCL-2 inhibitor, ABT-199, acquired resistance eventually occurs in many cases, which leads to CLL disease progression. Thus, understanding the mechanisms that mediate this relapse is important to design improved therapies. Herein, we report that cytokine IFN-γ, secreted by dysfunctional T cells, enhanced CLL cells resistance to ABT-199. IFN-γ stimulation significantly increased the expression of BCL-2, MCL-1 and BCL-xL. Blocking JAK1/2-STAT3 signaling pathway impaired the expression of these anti-apoptotic proteins after IFN-γ stimulation. The combination of ABT-199 with JAK1/2 inhibitor Ruxolitinib or STAT3 inhibitors Stattic and C188-9 increased malignant B cell death. In summary, we show that IFN-γ enhanced CLL cells resistance to ABT-199 at least in part by up-regulating BCL-2, MCL-1 and BCL-xL expression via JAK1/2-STAT3 pathway, and thus blocking this pathway with inhibitors increased ABT-199 efficiency to induce CLL cell apoptosis, suggesting a potential therapeutically relevant combination to overcome ABT-199 resistance.

Effects of compound sodium nitrophenol on microspore embryogenesis and plantlet regeneration in flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee).

Isolated microspore culture has been implemented in breeding programs to produce doubled haploid (DH) lines and thus accelerates the breeding process. However, low microspore embryogenesis frequency in flowering Chinese cabbage remains a key obstacle to the practical application of this technique. This study aimed to establish an efficient microspore culture protocol for flowering Chinese cabbage that would be applied for heterosis breeding. Microspores of five genotypes, 19AY05, 19AY06, 19AY10, 19AY12, and 19AY15, were successfully induced to produce embryos in NLN-13 medium. Microspores of two genotypes, 19AY05 and 19AY15, were cultivated in NLN-13 medium supplemented with different concentrations (0, 0.01, 0.05, 0.1, or 0.2 mg·L-1) of compound sodium nitrophenol (sodium nitrophenol, 5-nitrophenol) to enhance microspore embryogenesis and plant regeneration without an intervening callus phase. The results showed that 0.05 ~ 0.1 mg· L-1 sodium nitrophenol and 0.01 ~ 0.2 mg· L-1 of 5-nitrophenol significantly promoted the induction of microspore embryogenesis of two genotypes, and the best concentrations required for different genotypes are different. Moreover, 0.1 mg· L-1 sodium nitrophenol can significantly increase the plant regeneration rate of the two genetypes. The 5-nitrophenol at 0.01 mg·L-1 significantly increased rate of embryos directly convert to plant in 19AY15. In addition, the average doubled haploid rates in the five genotypes were close to 63%. Horticultural traits of DH lines from 19AY05 were identified and all of them were self-incompatible lines. They showed a high uniformity and consistency that can be directly used for hybrid breeding. Furthermore, the hybrid combination was prepared with the selected DH lines and the Guangdong nucleus genic sterile line GMS019 to screen the excellent hybrid combination for the flowering Chinese cabbage breeding program. This method accelerates the application of microspore culture in hybrid breeding of flowering Chinese cabbage.

Dual Akt and Bcl-2 inhibition induces cell-type specific modulation of apoptotic and autophagic signaling in castration resistant prostate cancer cell lines.

BACKGROUND: Cancer cell survival depends on the cross-regulation between apoptosis and autophagy which share common signaling pathways including PI3K/Akt/mTOR and Bcl-2. The aim of this study was to elucidate the modulation patterns between apoptosis and autophagy following dual inhibition by Akt inhibitor erufosine and Bcl-2 inhibitor ABT-737 in castration-resistant prostate cancer (CRPC) cell lines, PC-3 (Bax+) and DU-145 (Bax-). METHODS AND RESULTS: Cell cycle progression, apoptotic and autophagic signaling were examined by flow cytometry, multi-caspase assay, Hoechst staining, acridine orange staining of acidic vesicular organelles (AVOs), qRT-PCR and Western Blot. Dual inhibition increased G2/M arrest in PC-3 and DU-145, but not in the healthy prostate epithelium cells, PNT-1A. Only in PC-3, dual inhibition induced synergistic apoptotic and additive autophagic effects. In DU-145 and PNT-1A cells, ABT-737 did not display any remarkable effect on multicaspase activity and erufosine and ABT-737, neither alone nor in combination induced AVOs. By dual inhibition, AKT, BCL-2 and NF-κB gene expressions were downregulated in PC-3, both ATG-5 and BECLIN-1 gene expressions were upregulated in DU-145 but Beclin-1 protein expression was substantially reduced in both CRPC cells. Dual inhibition-induced synergistic multicaspase activation in PC-3 degrades and disrupts autophagic activity of Beclin-1, enhancing caspase-dependent apoptosis. However, in DU-145, following dual inhibition, rate of multicaspase induction and apoptosis are lower but autophagy is completely abolished despite markedly increased BECLIN-1 gene expression. CONCLUSION: In conclusion, antineoplastic drug combinations may display cell-type specific modulation of apoptotic and autophagic signaling and lack of protective autophagy may not necessarily indicate increased chemotherapeutic sensitivity in heterogenous tumor subpopulations.

Inhibitors of Anti-apoptotic Bcl-2 Family Proteins Exhibit Potent and Broad-Spectrum Anti-mammarenavirus Activity via Cell Cycle Arrest at G0/G1 Phase.

Targeting host factors is a promising strategy to develop broad-spectrum antiviral drugs. Drugs targeting anti-apoptotic Bcl-2 family proteins that were originally developed as tumor suppressors have been reported to inhibit multiplication of different types of viruses. However, the mechanisms whereby Bcl-2 inhibitors exert their antiviral activity remain poorly understood. In this study, we have investigated the mechanisms by which obatoclax (OLX) and ABT-737 Bcl-2 inhibitors exhibited a potent antiviral activity against the mammarenavirus lymphocytic choriomeningitis virus (LCMV). OLX and ABT-737 potent anti-LCMV activity was not associated with their proapoptotic properties but rather with their ability to induce cell arrest at the G0/G1 phase. OLX- and ABT-737-mediated inhibition of Bcl-2 correlated with reduced expression levels of thymidine kinase 1 (TK1), cyclin A2 (CCNA2), and cyclin B1 (CCNB1) cell cycle regulators. In addition, small interfering RNA (siRNA)-mediated knockdown of TK1, CCNA2, and CCNB1 resulted in reduced levels of LCMV multiplication. The antiviral activity exerted by Bcl-2 inhibitors correlated with reduced levels of viral RNA synthesis at early times of infection. Importantly, ABT-737 exhibited moderate efficacy in a mouse model of LCMV infection, and Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our results suggest that Bcl-2 inhibitors, actively being explored as anticancer therapeutics, might be repositioned as broad-spectrum antivirals. IMPORTANCE Antiapoptotic Bcl-2 inhibitors have been shown to exert potent antiviral activities against various types of viruses via mechanisms that are currently poorly understood. This study has revealed that Bcl-2 inhibitors' mediation of cell cycle arrest at the G0/G1 phase, rather than their proapoptotic activity, plays a critical role in blocking mammarenavirus multiplication in cultured cells. In addition, we show that Bcl-2 inhibitor ABT-737 exhibited moderate antimammarenavirus activity in vivo and that Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our results suggest that Bcl-2 inhibitors, actively being explored as anticancer therapeutics, might be repositioned as broad-spectrum antivirals.

A375 melanoma cells are sensitized to cisplatin-induced toxicity by a synthetic nitro-flavone derivative 2-(4-Nitrophenyl)-4H-chromen-4-one through inhibition of PARP1.

BACKGROUND: Cisplatin has been extensively used in therapeutics for its broad-spectrum anticancer activity and frequently used for the treatment of solid tumors. However, it presents several side-effects and several cancers develop resistance. Combination therapy of cisplatin with poly (ADP-ribose) polymerase 1 (PARP1) inhibitors has been effective in increasing its efficacy at lower doses. METHODS AND RESULTS: In this work, we have shown that the nitro-flavone derivative, 2-(4-Nitrophenyl)-4H-chromen-4-one (4NCO), can improve the sensitivity of cancer cells to cisplatin through inhibition of PARP1. The effect of 4NCO on cisplatin toxicity was studied through combination therapy in both exponential and density inhibited A375 melanoma cells. Combination index (CI) was determined from isobologram analysis. The mechanism of cell killing was assessed by lactate dehydrogenase (LDH) assay. Temporal nicotinamide adenine dinucleotide (NAD+) assay was done to show the inhibition of PARP1. We also performed in silico molecular modeling studies to know the binding mode of 4NCO to a modeled PARP1-DNA complex containing cisplatin-crosslinked adduct. The results from both in silico and in cellulo studies confirmed that PARP1 inhibition by 4NCO was most effective in sensitizing A375 melanoma cells to cisplatin. Isobologram analysis revealed that 4NCO reduced cell viability both in exponential and density inhibited A375 cells synergistically. The combination led to cell death through apoptosis. CONCLUSION: The synthetic nitro-flavone derivative 4NCO effectively inhibited the important nuclear DNA repair enzyme PARP1 and therefore, could complement the DNA-damaging anticancer drug cisplatin in A375 cells and thus, could act as a potential adjuvant to cisplatin in melanoma therapy.

Antibacterial Activity of a Promising Antibacterial Agent: 22-(4-(2-(4-Nitrophenyl-piperazin-1-yl)-acetyl)-piperazin-1-yl)-22-deoxypleuromutilin.

A novel pleuromutilin derivative, 22-(4-(2-(4-nitrophenyl-piperazin-1-yl)-acetyl)-piperazin-1-yl)-22-deoxypleuromutilin (NPDM), was synthesized in our laboratory and proved excellent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). In this study, more methods were used to further study its preliminary pharmacological effect. The antibacterial efficacy and toxicity of NPDM were evaluated using tiamulin as the reference drug. The in vitro antibacterial activity study showed that NPDM is a potent bactericidal agent against MRSA that induced time-dependent growth inhibition and a concentration-dependent post-antibiotic effect (PAE). Toxicity determination showed that the cytotoxicity of NPDM was slightly higher than that of tiamulin, but the acute oral toxicity study proved that NPDM was a low-toxic compound. In an in vivo antibacterial effect study, NPDM exhibited a better therapeutic effect than tiamulin against MRSA in a mouse thigh infection model as well as a mouse systemic infection model with neutropenia. The 50% effective dose (ED50) of NPDM in a Galleria mellonella infection model was 50.53 mg/kg. The pharmacokinetic properties of NPDM were also measured, which showed that NPDM was a rapid elimination drug in mice.

BH3-only protein expression determines hepatocellular carcinoma response to sorafenib-based treatment.

Hepatocellular carcinoma (HCC) represents a global health challenge with limited therapeutic options. Anti-angiogenic immune checkpoint inhibitor-based combination therapy has been introduced for progressed HCC, but improves survival only in a subset of HCC patients. Tyrosine-kinase inhibitors (TKI) such as sorafenib represent an alternative treatment option but have only modest efficacy. Using different HCC cell lines and HCC tissues from various patients reflecting HCC heterogeneity, we investigated whether the sorafenib response could be enhanced by combination with pro-apoptotic agents, such as TNF-related apoptosis-inducing ligand (TRAIL) or the BH3-mimetic ABT-737, which target the death receptor and mitochondrial pathway of apoptosis, respectively. We found that both agents could enhance sorafenib-induced cell death which was, however, dependent on specific BH3-only proteins. TRAIL augmented sorafenib-induced cell death only in NOXA-expressing HCC cells, whereas ABT-737 enhanced the sorafenib response also in NOXA-deficient cells. ABT-737, however, failed to augment sorafenib cytotoxicity in the absence of BIM, even when NOXA was strongly expressed. In the presence of NOXA, BIM-deficient HCC cells could be in turn strongly sensitized for cell death induction by the combination of sorafenib with TRAIL. Accordingly, HCC tissues sensitive to apoptosis induction by sorafenib and TRAIL revealed enhanced NOXA expression compared to HCC tissues resistant to this treatment combination. Thus, our results suggest that BH3-only protein expression determines the treatment response of HCC to different sorafenib-based drug combinations. Individual profiling of BH3-only protein expression might therefore assist patient stratification to certain TKI-based HCC therapies.

Verticillin A increases the BIMEL/MCL-1 ratio to overcome ABT-737-resistance in human colon cancer cells by targeting the MEK/ERK pathway.

ABT-737, a small molecule BH-3 mimetic, is less effective against human colon cancers due to its resistance. Verticillin A is a natural compound, which was previously purified from verticillium-infected mushrooms. Hence, we aimed at overcoming the ABT737 resistance observed in CRC tumors by combining Verticillin A with ABT-737 and figuring out the potential mechanism. In this study, we observed that Verticillin A could sensitize colon cancer to ABT-737-induced cell death through induction of mitochondrial-dependent apoptosis. Verticillin A could significantly increase the BIMEL/MCL-1 ratio to overcome ABT737 resistance through the suppression of the MEK/ERK pathway. In addition, up-regulation of BIM protein levels to activate BAX translocation results in apoptosis induction. Altogether, our work suggested the potential application of Verticillin A as a MEK inhibitor in BH3-mimetic-based therapy.

Curcumin at Low Doses Potentiates and at High Doses Inhibits ABT-737-Induced Platelet Apoptosis.

Curcumin is a natural bioactive component derived from the turmeric plant Curcuma longa, which exhibits a range of beneficial activities on human cells. Previously, an inhibitory effect of curcumin on platelets was demonstrated. However, it is unknown whether this inhibitory effect is due to platelet apoptosis or procoagulant platelet formation. In this study, curcumin did not activate caspase 3-dependent apoptosis of human platelets, but rather induced the formation of procoagulant platelets. Interestingly, curcumin at low concentration (5 µM) potentiated, and at high concentration (50 µM) inhibited ABT-737-induced platelet apoptosis, which was accompanied by inhibition of ABT-737-mediated thrombin generation. Platelet viability was not affected by curcumin at low concentration and was reduced by 17% at high concentration. Furthermore, curcumin-induced autophagy in human platelets via increased translocation of LC3I to LC3II, which was associated with activation of adenosine monophosphate (AMP) kinase and inhibition of protein kinase B activity. Because curcumin inhibits P-glycoprotein (P-gp) in cancer cells and contributes to overcoming multidrug resistance, we showed that curcumin similarly inhibited platelet P-gp activity. Our results revealed that the platelet inhibitory effect of curcumin is mediated by complex processes, including procoagulant platelet formation. Thus, curcumin may protect against or enhance caspase-dependent apoptosis in platelets under certain conditions.

GSK-3ß Can Regulate the Sensitivity of MIA-PaCa-2 Pancreatic and MCF-7 Breast Cancer Cells to Chemotherapeutic Drugs, Targeted Therapeutics and Nutraceuticals.

Glycogen synthase kinase-3 (GSK-3) is a regulator of signaling pathways. KRas is frequently mutated in pancreatic cancers. The growth of certain pancreatic cancers is KRas-dependent and can be suppressed by GSK-3 inhibitors, documenting a link between KRas and GSK-3. To further elucidate the roles of GSK-3ß in drug-resistance, we transfected KRas-dependent MIA-PaCa-2 pancreatic cells with wild-type (WT) and kinase-dead (KD) forms of GSK-3ß. Transfection of MIA-PaCa-2 cells with WT-GSK-3ß increased their resistance to various chemotherapeutic drugs and certain small molecule inhibitors. Transfection of cells with KD-GSK-3ß often increased therapeutic sensitivity. An exception was observed with cells transfected with WT-GSK-3ß and sensitivity to the BCL2/BCLXL ABT737 inhibitor. WT-GSK-3ß reduced glycolytic capacity of the cells but did not affect the basal glycolysis and mitochondrial respiration. KD-GSK-3ß decreased both basal glycolysis and glycolytic capacity and reduced mitochondrial respiration in MIA-PaCa-2 cells. As a comparison, the effects of GSK-3 on MCF-7 breast cancer cells, which have mutant PIK3CA, were examined. KD-GSK-3ß increased the resistance of MCF-7 cells to chemotherapeutic drugs and certain signal transduction inhibitors. Thus, altering the levels of GSK-3ß can have dramatic effects on sensitivity to drugs and signal transduction inhibitors which may be influenced by the background of the tumor.

Is PF-00835231 a Pan-SARS-CoV-2 Mpro Inhibitor? A Comparative Study.

The COVID-19 outbreak continues to spread worldwide at a rapid rate. Currently, the absence of any effective antiviral treatment is the major concern for the global population. The reports of the occurrence of various point mutations within the important therapeutic target protein of SARS-CoV-2 has elevated the problem. The SARS-CoV-2 main protease (Mpro) is a major therapeutic target for new antiviral designs. In this study, the efficacy of PF-00835231 was investigated (a Mpro inhibitor under clinical trials) against the Mpro and their reported mutants. Various in silico approaches were used to investigate and compare the efficacy of PF-00835231 and five drugs previously documented to inhibit the Mpro. Our study shows that PF-00835231 is not only effective against the wild type but demonstrates a high affinity against the studied mutants as well.

ABT­737, a Bcl­2 family inhibitor, has a synergistic effect with apoptosis by inducing urothelial carcinoma cell necroptosis.

ABT­737 is a recently reported inhibitor of members of the Bcl­2 family of apoptosis regulators. However, to the best of our knowledge, its necroptosis­inducing function in bladder cancer has not yet been researched. Thus, the present study aimed to investigate whether this Bcl­2 family inhibitor can induce both apoptosis and necroptosis of urothelial carcinoma cells. The proliferation and survival of urothelial carcinoma cell lines treated with a combination of both Z­VAD­FMK as a pan­caspase inhibitor and ABT­737 were assessed in vitro. Z­DNA binding protein 1 (ZBP1), receptor­interacting protein (RIP)1 and RIP3 were knocked down using small interfering RNA in urothelial carcinoma cell lines. The protein expression levels of ZBP1, RIP1 and RIP3 following cell transfection were measured via western blot analysis. Cell viability was determined using an MTT assay. Cell invasion was examined using cell invasion assays. The expression levels of necroptosis­related proteins, high mobility group box 1, ZBP1, mixed­lineage kinase domain­like protein (MLKL) and RIP3, were measured via western blotting. It was found that ABT­737 inhibited the proliferation and invasion of bladder cancer cells by inducing cell necrosis. The data demonstrated that ZBP1 and RIP3 have main roles in the cell necrosis induced by ABT­737. In addition, RIP3 and ZBP1, without interacting with RIP1, directly induced MLKL­mediated programmed cell necrosis. Thus, understanding how urothelial carcinoma cells react to Bcl­2 family inhibitors may accelerate the discovery of drugs to treat bladder cancer.

GNA13 regulates BCL2 expression and the sensitivity of GCB-DLBCL cells to BCL2 inhibitors in a palmitoylation-dependent manner.

GNA13, encoding one of the G protein alpha subunits of heterotrimeric G proteins that transduce signals of G protein-coupled receptors (GPCR), is frequently mutated in germinal center B-cell-like diffuse large B-cell lymphoma (GCB-DLBCL) with poor prognostic outcomes. Due to the "undruggable" nature of GNA13, targeted therapy for these patients is not available. In this study, we found that palmitoylation of GNA13 not only regulates its plasma membrane localization, but also regulates GNA13's stability. It is essential for the tumor suppressor function of GNA13 in GCB-DLBCL cells. Interestingly, GNA13 negatively regulates BCL2 expression in GCB-DLBCL cells in a palmitoylation-dependent manner. Consistently, BCL2 inhibitors were found to be effective in killing GNA13-deficient GCB-DLBCL cells in a cell-based chemical screen. Furthermore, we demonstrate that inactivating GNA13 by targeting its palmitoylation enhanced the sensitivity of GCB-DLBCL to the BCL2 inhibitor. These studies indicate that the loss-of-function mutation of GNA13 is a biomarker for BCL2 inhibitor therapy of GCB-DLBCL and that GNA13 palmitoylation is a potential target for combination therapy with BCL2 inhibitors to treat GCB-DLBCL with wild-type GNA13.

NOXA upregulation by the prohibitin-binding compound fluorizoline is transcriptionally regulated by integrated stress response-induced ATF3 and ATF4.

Fluorizoline is a new synthetic molecule that induces p53-independent apoptosis, in several tumor cell lines and in primary leukemia cells, by selectively targeting prohibitins (PHBs). In this study, we describe how fluorizoline induces BCL-2 homology 3-only protein NOXA, without modulating the protein levels of anti-apoptotic B-cell lymphoma-2 (BCL-2) family members prior to caspase activation, as well as how it synergizes with the BCL-2 and BCL-XL inhibitor ABT-737 to induce apoptosis. Interestingly, fluorizolinetreatment triggers the activation of the integrated stress response (ISR) in HeLa and HAP1 cells, with increased eukaryotic translation initiation factor 2α phosphorylation, and induction of ATF3, ATF4, and CHOP. Moreover, PHB downregulation induces similar ISR activation and apoptosis as with fluorizoline treatment. In addition, we studied the essential role of the pro-apoptotic protein NOXA in fluorizoline-induced apoptosis and we describe its mechanism of induction in HeLa and HAP1 cells. Moreover, we identified ATF3 and ATF4 as the transcription factors that bind to NOXA promoter upon fluorizoline treatment. Furthermore, using ATF3 and ATF4 CRISPR HeLa and HAP1 cells, we confirmed that both factors mediate the induction of NOXA and apoptosis by fluorizoline. In conclusion, fluorizoline treatment triggers the activation of the ISR that results in the induction of ATF3 and ATF4, important regulators of NOXA transcription in fluorizoline-induced apoptosis.