Poly ADP-ribose Polymerase Cleavage Research Articles

Vitamin C alleviates hyperglycemic stress in retinal pigment epithelial cells.

Retinal pigment epithelial cells (RPECs) are a type of retinal cells that structurally and physiologically support photoreceptors. However, hyperglycemia has been shown to play a critical role in the progression of diabetic retinopathy (DR), which is one of the leading causes of vision impairment. In the diabetic eye, the high glucose environment damages RPECs via the induction of oxidative stress, leading to the release of excess reactive oxygen species (ROS) and triggering apoptosis. In this study, we aim to investigate the antioxidant mechanism of Vitamin C in reducing hyperglycemia-induced stress and whether this mechanism can preserve the function of RPECs. ARPE-19 cells were treated with high glucose in the presence or absence of Vitamin C. Cell viability was measured by MTT assay. Cleaved poly ADP-ribose polymerase (PARP) was used to identify apoptosis in the cells. ROS were detected by the DCFH-DA reaction. The accumulation of sorbitol in the aldose reductase (AR) polyol pathway was determined using the sorbitol detection assay. Primary mouse RPECs were isolated from adult mice and identified by Rpe65 expression. The mitochondrial damage was measured by mitochondrial membrane depolarization. Our results showed that high glucose conditions reduce cell viability in RPECs while Vitamin C can restore cell viability, compared to the vehicle treatment. We also demonstrated that Vitamin C reduces hyperglycemia-induced ROS production and prevents cell apoptosis in RPECs in an AR-independent pathway. These results suggest that Vitamin C is not only a nutritional necessity but also an adjuvant that can be combined with AR inhibitors for alleviating hyperglycemic stress in RPECs.

MK591 (Quiflapon), a 5-lipoxygenase inhibitor, kills pancreatic cancer cells via downregulation of protein kinase C-epsilon.

Pancreatic tumors and cell lines derived from them exhibit elevated expression of 5-lipoxygenase (5-Lox), whereas non-tumor glands or normal cells do not exhibit this overexpression. Arachidonic acid stimulates pancreatic cancer cell growth via metabolic conversion through the 5-Lox pathway, and inhibition of 5-Lox activity decreases the viability of pancreatic cancer cells. However, the downstream signaling mechanisms through which 5-Lox exerts its effects on the survival of pancreatic cancer cells remain to be elucidated. The effects of 5-Lox inhibition on cell proliferation, apoptosis, and invasive potential were investigated in pancreatic cancer cells. The protein expression was analyzed by Western blot. Apoptosis was analyzed by Annexin-V binding assay and by detecting the degradation of chromatin-DNA to nucleosomal fragments. The protein kinase C-epsilon (PKCε) activity was measured by an immunoprecipitation-kinase assay. The in vivo effects of MK591 were evaluated in pancreatic tumor xenograft model. MK591, a specific inhibitor of 5-Lox activity, killed pancreatic cancer cells via induction of apoptosis, involving externalization of phosphatidylserine, cleavage of PARP (poly-ADP ribose polymerase) and degradation of chromatin DNA to nucleosomes. MK591 effectively blocked in vitro invasion and soft-agar colony formation by pancreatic cancer cells and decreased pancreatic tumor growth in nude mice xenografts. Furthermore, inhibition of 5-Lox downregulated K-Ras and inhibited phosphorylation of c-Raf and ERKs. Interestingly, 5-Lox inhibition induced apoptosis in pancreatic cancer cells without the inhibition of Akt but the protein level of PKCε was dramatically downregulated. Furthermore, inhibition of 5-Lox decreased the phosphorylation of Stat3 at Serine-727. Pre-treatment of pancreatic cancer cells with peptide activators of PKCε prevented apoptosis induced by 5-Lox inhibition, suggesting that the mechanism by which 5-Lox inhibition causes cell death in pancreatic cancer involves downregulation of PKCε. The combination of low doses of MK591 and gemcitabine synergistically reduced the oncogenic phenotype and killed pancreatic cancer cells by inducing apoptosis. These findings indicate that inhibition of 5-Lox interrupts an Akt-independent, PKCε-dependent survival mechanism in pancreatic cancer cells and suggest that metabolism of arachidonic acid through the 5-Lox pathway plays an integral part in the survival of pancreatic cancer cells via signaling through PKCε, an oncogenic, pro-survival serine/threonine kinase.

Cymensifin A: a promising pharmaceutical candidate to defeat lung cancer via cellular reactive oxygen species-mediated apoptosis.

Background: The upgrade of natural products for cancer treatment is essential since current anticancer drugs still pose severe side effects. Cymensifin A (Cym A) isolated from an orchid Cymbidium ensifolium has shown its potential to induce the death of several cancer cells; however, its underlying molecular mechanisms are hitherto unknown. Methods: Here, we conducted a set of in vitro preliminary tests to assess the cytotoxic effects of Cym A on non-small-cell lung cancer (NSCLC) cells (A549, H23, H292, and H460). A flow cytometry system and Western blot analyses were employed to unveil molecular mechanisms underlying cancer cell apoptosis caused by Cym A. Results: Cym A at 25-50μM caused the death of all NSCLC cells tested, and its cytotoxicity was comparable to cisplatin, a currently used anticancer drug. The compound induced apoptosis of all NSCLC cells in a dose-dependent manner (5-50μM), proven by flow cytometry, but H460 cells showed more resistance compared to other cells tested. Cym A-treated H460 cells demonstrated increased reactive oxygen species (ROS) and downregulated antioxidants (catalase, superoxide dismutase, and thioredoxin). The compound also upregulated the tumor suppressor P53 and the pro-apoptotic protein BAX but downregulated pro-survival proteins (BCL-2 and MCL-1) and deactivated survival signals (AKT and ERK) in H460 cells. Cym A was proven to trigger cellular ROS formation, but P53 and BAX were 2-fold more activated by Cym A compared to those treated with hydrogen peroxide. Our findings also supported that Cym A exerted its roles in the downregulation of nuclear factor erythroid 2-related factor 2 (a regulator of cellular antioxidant activity) and the increased levels of cleaved poly (ADP-ribose) polymerase (PARP) and cleaved caspase 3/7 during apoptosis. Conclusion: We propose that Cym A induces lung cancer cell death via ROS-mediated apoptosis, while the modulation of cellular ROS/antioxidant activity, the upregulation of P53 and BAX, the downregulation or deactivation of BCL-2, MCL-1, AKT, and ERK, and the increased cleavage of PARP and caspase 3/7, were the elucidated underlying molecular mechanisms of this phytochemical. The compound can be a promising candidate for future anticancer drug development.

Repurposing the diuretic benzamil as an anti-osteosarcoma agent that acts by suppressing integrin/FAK/STAT3 signalling and compromising mitochondrial function.

Osteosarcoma is the most common primary bone malignancy among children and adolescents. We investigated whether benzamil, an amiloride analogue and sodium-calcium exchange blocker, may exhibit therapeutic potential for osteosarcoma in vitro. MG63 and U2OS cells were treated with benzamil for 24 hours. Cell viability was evaluated with the MTS/PMS assay, colony formation assay, and flow cytometry (forward/side scatter). Chromosome condensation, the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, cleavage of poly-ADP ribose polymerase (PARP) and caspase-7, and FITC annexin V/PI double staining were monitored as indicators of apoptosis. Intracellular calcium was detected by flow cytometry with Fluo-4 AM. The phosphorylation and activation of focal adhesion kinase (FAK) and signal transducer and activator of transcription 3 (STAT3) were measured by western blot. The expression levels of X-linked inhibitor of apoptosis protein (XIAP), B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra large (Bcl-xL), SOD1, and SOD2 were also assessed by western blot. Mitochondrial status was assessed with tetramethylrhodamine, ethyl ester (TMRE), and intracellular adenosine triphosphate (ATP) was measured with BioTracker ATP-Red Live Cell Dye. Total cellular integrin levels were evaluated by western blot, and the expression of cell surface integrins was assessed using fluorescent-labelled antibodies and flow cytometry. Benzamil suppressed growth of osteosarcoma cells by inducing apoptosis. Benzamil reduced the expression of cell surface integrins α5, αV, and β1 in MG63 cells, while it only reduced the expression of αV in U2OS cells. Benzamil suppressed the phosphorylation and activation of FAK and STAT3. In addition, mitochondrial function and ATP production were compromised by benzamil. The levels of anti-apoptotic proteins XIAP, Bcl-2, and Bcl-xL were reduced by benzamil. Correspondingly, benzamil potentiated cisplatin- and methotrexate-induced apoptosis in osteosarcoma cells. Benzamil exerts anti-osteosarcoma activity by inducing apoptosis. In terms of mechanism, benzamil appears to inhibit integrin/FAK/STAT3 signalling, which triggers mitochondrial dysfunction and ATP depletion.

Design, Synthesis, and Anti-Leukemic Evaluation of a Series of Dianilinopyrimidines by Regulating the Ras/Raf/MEK/ERK and STAT3/c-Myc Pathways.

Although the long-term survival rate for leukemia has made significant progress over the years with the development of chemotherapeutics, patients still suffer from relapse, leading to an unsatisfactory outcome. To discover the new effective anti-leukemia compounds, we synthesized a series of dianilinopyrimidines and evaluated the anti-leukemia activities of those compounds by using leukemia cell lines (HEL, Jurkat, and K562). The results showed that the dianilinopyrimidine analog H-120 predominantly displayed the highest cytotoxic potential in HEL cells. It remarkably induced apoptosis of HEL cells by activating the apoptosis-related proteins (cleaved caspase-3, cleaved caspase-9 and cleaved poly ADP-ribose polymerase (PARP)), increasing apoptosis protein Bad expression, and decreasing the expression of anti-apoptotic proteins (Bcl-2 and Bcl-xL). Furthermore, it induced cell cycle arrest in G2/M; concomitantly, we observed the activation of p53 and a reduction in phosphorylated cell division cycle 25C (p-CDC25C) / Cyclin B1 levels in treated cells. Additionally, the mechanism study revealed that H-120 decreased these phosphorylated signal transducers and activators of transcription 3, rat sarcoma, phosphorylated cellular RAF proto-oncogene serine / threonine kinase, phosphorylated mitogen-activated protein kinase kinase, phosphorylated extracellular signal-regulated kinase, and cellular myelocytomatosis oncogene (p-STAT3, Ras, p-C-Raf, p-MEK, p-MRK, and c-Myc) protein levels in HEL cells. Using the cytoplasmic and nuclear proteins isolation assay, we found for the first time that H-120 can inhibit the activation of STAT3 and c-Myc and block STAT3 phosphorylation and dimerization. Moreover, H-120 treatment effectively inhibited the disease progression of erythroleukemia mice by promoting erythroid differentiation into the maturation of erythrocytes and activating the immune cells. Significantly, H-120 also improved liver function in erythroleukemia mice. Therefore, H-120 may be a potential chemotherapeutic drug for leukemia patients.

Sequence-dependent effects of hematoporphyrin derivatives (HPD) photodynamic therapy and cisplatin on lung adenocarcinoma cells

BackgroundHematoporphyrin derivatives (HPD)-Photodynamic therapy (PDT) in combination with cisplatin (DDP) is an effective anticancer strategy. However, whether the order of combination affects efficacy has not been studied. MethodsThe human lung adenocarcinoma (LUAD) A549 cells were used as the study subjects. After A549 cells were treated with a single medication (PDT/DDP) or a sequential combination (PDT + DDP / DDP + PDT), the cell viability was assayed using the cell counting kit-8 method. Hoechst staining, Annexin-V/propidium iodide (PI) double staining, western blotting, and a real-time quantitative polymerase chain reaction (RT-qPCR) were performed to examine the mechanisms behind the combined effects. ResultsA synergistic impact between HPD-PDT and DDP was found. The cell viability in the PDT+DDP group was significantly lower than in the DDP+PDT group. A significant apoptotic profile and a high apoptotic rate were seen in the PDT + DDP group. The western blot showed that the expression levels of Bcl2-associated x(Bax) and cleaved-poly ADP-ribose polymerase (PARP) increased, and those of B-cell lymphoma-2 (Bcl-2) and Caspase-9 decreased in the PDT + DDP group. At the same time, the RT-qPCR revealed the upregulation of Bax and PARP mRNA and the downregulation of Bcl-2 and Caspase-9 mRNA. ConclusionThe order of the combination therapy (PDT + DDP / DDP + PDT) was important. The HPD-PDT followed by DDP significantly inhibited LUAD cell viability, which may be related to the mitochondrial apoptotic pathway.

Pharmacological p38 MAPK inhibitor SB203580 enhances AML stem cell line KG1a chemosensitivity to daunorubicin by promoting late apoptosis, cell growth arrest in S-phase, and miR-328-3p upregulation

Acute myeloid leukaemia (AML) is characterized by uncontrolled proliferation of myeloid progenitor cells and impaired maturation, leading to immature cell accumulation in the bone marrow and bloodstream, resulting in hematopoietic dysfunction. Chemoresistance, hyperactivity of survival pathways, and miRNA alteration are major factors contributing to treatment failure and poor outcomes in AML patients. This study aimed to investigate the impact of the pharmacological p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 on the chemoresistance potential of AML stem cell line KG1a to the therapeutic drug daunorubicin (DNR). KG1a and chemosensitive leukemic HL60 cells were treated with increasing concentrations of DNR. Cell Titer-Glo®, flow cytometry, phosphokinase and protein arrays, Western blot technology, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were employed for assessment of cell viability, half-maximal inhibitory concentration (IC50) determination, apoptotic status detection, cell cycle analysis, apoptosis-related protein and gene expression monitoring. Confocal microscopy was used to visualize caspase and mitochondrial permeability transition pore (mPTP) activities. Exposed at various incubation times, higher DNR IC50 values were determined for KG1a cells than for HL60 cells, confirming KG1a cell chemoresistance potential. Exposed to DNR, late apoptosis induction in KG1a cells was enhanced after SB203580 pretreatment, defined as the combination treatment. This enhancement was confirmed by increased cleavage of poly(ADP-ribose) polymerase, caspase-9, caspase-3, and augmented caspase-3/-7 and mPTP activities in KG1a cells upon combination treatment, compared to DNR. Using phosphokinase and apoptosis protein arrays, the combination treatment decreased survival Akt phosphorylation and anti-apoptotic Bcl-2 expression levels in KG1a cells while increasing the expression levels of the tumor suppressor p53 and cyclin-dependent kinase inhibitor p21, compared to DNR. Cell cycle analysis revealed KG1a cell growth arrest in G2/M-phase caused by DNR, while combined treatment led to cell growth arrest in S-phase, mainly associated with cyclin B1 expression levels. Remarkably, the enhanced KG1a cell sensitivity to DNR after SB203580 pretreatment was associated with an increased upregulation of miR-328-3p and slight downregulation of miR-26b-5p, compared to DNR effect. Altogether, these findings could contribute to the development of a new therapeutic strategy by targeting the p38 MAPK pathway to improve treatment outcomes in patients with refractory or relapsed AML.

Abstract 1912: Targeting Trop-2 with sacituzumab govitecan (IMMU-132) suppresses the tumor growth in diffuse large B cell lymphoma

Abstract Introduction: Sacituzumab govitecan (SG), also termed IMMU-132, is an antibody-drug conjugate, linking an anti-Trop-2 antibody and the topoisomerase I inhibitor, SN-38. Clinically, SG has shown promising therapeutic effects in patients with human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer. However, the efficacy of SG in diffuse large B-cell lymphoma (DLBCL) remains unexplored. In this study, we sought to determine whether SG can efficiently inhibit cell proliferation and tumor growth of DLBCL by using both in vitro and in vivo models. Methods: We examined the cytotoxicity of SG in the activated B-cell like (ABC)-subtype-DLBCL cell lines, Riva (Ri-1), HBL-1, and SU-DHL-2, and the germinal center B-cell (GCB)-subtype-DLBCL cell lines, SU-DHL-4, SU-DHL-5, and SU-DHL-6 by MTT assay. We assessed the distributions of cell cycle phase and apoptosis in the aforementioned DLBCL cell lines treated with SG for 24 h or 48 h using flow cytometry and Annexin V staining. We confirmed apoptosis (cleavage of poly ADP-ribose polymerase [PARP] and caspase-3) and detected DNA damage (γH2AX and 53BP1)-related protein expression in SU-DHL-2 and SU-DHL-6 cell lines treated with SG at various concentrations for 48 h using western blotting, compared to untreated cells. To examine the anti-tumor effect of SG in vivo, NOD-SCID nude mice of Riva-xenograft mouse models were randomized into a control group and an SG-treated group. Results: Flow cytometry and western blotting revealed overexpression of Trop-2 on the surface of untreated ABC-DLBCL and GCB-DLBCL cell lines. In this study, we showed that SG treatment reduced cell viability in all six DLBCL cell lines with nanomolar IC50 values. Exposing SG to DLBCL cells significantly increased cell cycle arrest at the G2/M and sub-G1 phases of all six DLBCL cell lines. Annexin V staining revealed that SG induced apoptosis in all six DLBCL cell lines. Western blotting confirmed apoptosis induction by showing a dose-dependent upregulation of cleaved PARP and cleaved caspases-3 by SG treatment, along with decreased expression of survivin in both SU-DHL-2 and SU-DHL-6 cells. SG also upregulated the expression of γH2AX and 53BP1 in both SU-DHL-2 and SU-DHL-6 cells. Furthermore, we found that tumor growth of Riva cells in vivo was significantly reduced by treatment with SG when compared with untreated control. Conclusion: SG suppresses tumor growth of ABC-DLBCL and GCB-DLBCL cells in both in vitro and in vivo models by inducing cell cycle arrest, apoptosis, and DNA damage. These findings provide a rationale for transitioning SG to clinical trials for treating patients with relapsed or refractory DLBCL. Citation Format: Jakie Ting, Ming-Feng Wei, Hsiao-Wei Lee, Keng-Hsueh Lan, Ann-Lii Cheng, Sung-Hsin Kuo. Targeting Trop-2 with sacituzumab govitecan (IMMU-132) suppresses the tumor growth in diffuse large B cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1912.

Abstract A013: Auranofin induces lethality driven by reactive oxygen species in high-grade serous ovarian cancer cells

Abstract High-grade serous ovarian cancer (HGSOC) accounts for 70% of ovarian cancer cases in the clinical setting; the survival rate for this disease remains remarkably low due to the lack of a long-term consolidation therapy following standard platinum-based chemotherapy. There is minimal improvement in the survival rate of this disease due to the development of resistance to platinum drugs as the disease recurs. The purpose of this study was to develop a novel treatment modality against HGSOC using the gold complex auranofin (AF). AF primarily functions as a pro-oxidant agent through the inhibition of thioredoxin reductase (TrxR), an antioxidant enzyme that is overexpressed in ovarian cancer. We explored the effect of AF on TrxR activity and various mechanisms of cytotoxicity using HGSOC cells that are clinically sensitive (PEO1) or resistant (PEO4) to platinum. We treated PEO1 cells and PEO4 cells with AF for 72 hours and analyzed its short-term toxicity by assessing cell viability and DNA content by flow cytometry, and the long-term anti-proliferative capacity using colony formation assays. We also assessed the mechanisms of cell death induced by AF including the activation of mechanisms of apoptosis, activation of executor caspases -3 and -7, cleavage of poly-(ADP-ribose) polymerase (PARP), changes in the mitochondrial membrane potential (MMP), and DNA damage. In addition, we studied the interaction between AF and another pro-oxidant, L-buthionine sulfoximine (L-BSO), an anti-glutathione (GSH) agent. We demonstrate that AF potently inhibits the activity of TrxR at non-cytotoxic concentrations while reducing the viability of HGSOC cells regardless of their sensitivities to platinum. We showed that AF induces accumulation of reactive oxygen species (ROS), triggers the depolarization of the mitochondrial membrane, and kills HGSOC cells by inducing apoptosis. AF-induced cell death and morphological effects were abrogated by the ROS scavenger, N-acetyl cysteine (NAC). In addition, the lethality of AF in HGSOC was associated with the activation of caspase-3/7, cleavage of PARP, and the generation of DNA damage, effects that were prevented by the ROS scavenger NAC. Finally, when AF and L-BSO were combined, we observed a synergistic lethality against HGSOC cells, which was mediated by a further increase in ROS with a decrease in the antioxidant, GSH. In summary, our results support the concept that AF can be used alone to kill HGSOC cells by causing ROS-mediated apoptosis. Furthermore, we demonstrate that a large depletion of antioxidants caused by combining AF and L-BSO is highly efficient in synergistically killing HGSOC cells. These findings suggest that the combination of AF and L-BSO is a potential option to develop a consolidation therapy against HGSOC, which can overcome the problem of platinum resistance during recurrent stages of the disease. Citation Format: Farah H. Abdalbari, Elvis M. Jaramillo, Benjamin N. Forgie, Estelle Tran, Alicia A. Goyeneche, Carlos M. Telleria. Auranofin induces lethality driven by reactive oxygen species in high-grade serous ovarian cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A013.

Integrated transcriptome and cell phenotype analysis suggest involvement of PARP1 cleavage, Hippo/Wnt, TGF-β and MAPK signaling pathways in ovarian cancer cells response to cannabis and PARP1 inhibitor treatment.

Introduction: Cannabis sativa is utilized mainly for palliative care worldwide. Ovarian cancer (OC) is a lethal gynecologic cancer. A particular cannabis extract fraction ('F7') and the Poly(ADP-Ribose) Polymerase 1 (PARP1) inhibitor niraparib act synergistically to promote OC cell apoptosis. Here we identified genetic pathways that are altered by the synergistic treatment in OC cell lines Caov3 and OVCAR3. Materials and methods: Gene expression profiles were determined by RNA sequencing and quantitative PCR. Microscopy was used to determine actin arrangement, a scratch assay to determine cell migration and flow cytometry to determine apoptosis, cell cycle and aldehyde dehydrogenase (ALDH) activity. Western blotting was used to determine protein levels. Results: Gene expression results suggested variations in gene expression between the two cell lines examined. Multiple genetic pathways, including Hippo/Wnt, TGF-β/Activin and MAPK were enriched with genes differentially expressed by niraparib and/or F7 treatments in both cell lines. Niraparib + F7 treatment led to cell cycle arrest and endoplasmic reticulum (ER) stress, inhibited cell migration, reduced the % of ALDH positive cells in the population and enhanced PARP1 cleavage. Conclusion: The synergistic effect of the niraparib + F7 may result from the treatment affecting multiple genetic pathways involving cell death and reducing mesenchymal characteristics.

Triphala extract Reduces Anti-apoptotic Protein (Mcl-1) Expression through the Activation of Caspase 3 in Hepatocellular Carcinoma Cells

Triphala is an equal mixture of the three myrobalans Terminalia chebula (Haritaki), Terminalia bellerica (Bibhitaki), and Phyllanthus emblica (Amalaki), according to the Ayurvedic Formulary of India. Triphala is an herbal supplement that is high in antioxidants and has a number of advantageous qualities. Numerous investigations conducted in recent years have shown that Triphala has anti-mutagenic and radioprotective properties. Therefore, the aim of the present study was to examine if an ethanolic extract of Triphala had any inhibitory effects on hepatocellular carcinoma cells (HCCs). Gas chromatography-mass spectrometry analysis was used to identify and quantify various phytochemicals in triphala extract (TE). Further, in two human liver cancer cells (HepG2 and Hep3B), TE was examined for its cytotoxic potential by [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Apoptosis was promoted by TE, which also suppressed the production of anti-apoptotic proteins and triggered poly-ADP ribose polymerase cleavage. Significantly, TE has a cytotoxic effect against liver cancer cells. The current study demonstrated that the ethanolic extract of triphala has anticancer properties against HCC. As a result, Triphala may offer a potential alternative to conventional therapy for cancer patients.

MHY446 induces apoptosis via reactive oxygen species-mediated endoplasmic reticulum stress in HCT116 human colorectal cancer cells

This study investigated the potential of a newly synthesized histone deacetylase (HDAC) inhibitor, MHY446, in inducing cell death in HCT116 colorectal cancer cells and compared its activity with that of suberoylanilide hydroxamic acid (SAHA), a well-known HDAC inhibitor. The results showed that MHY446 increased the acetylation of histones H3 and H4 and decreased the expression and activity of HDAC proteins in HCT116 cells. Additionally, MHY446 was confirmed to bind more strongly to HDAC1 than HDAC2 and inhibit its activity. In vivo experiments using nude mice revealed that MHY446 was as effective as SAHA in inhibiting HCT116 cell-grafted tumor growth. This study also evaluated the biological effects of MHY446 on cell survival and death pathways. The reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC) confirmed that ROS play a role in MHY446-induced cell death by reducing poly(ADP-ribose) polymerase cleavage. MHY446 also induced cell death via endoplasmic reticulum (ER) stress by increasing the expression of ER stress-related proteins. NAC treatment decreased the expression of ER stress-related proteins, indicating that ROS mediate ER stress as an upstream signaling pathway and induce cell death. While MHY446 did not exhibit superior HDAC inhibition efficacy compared to SAHA, it is anticipated to provide innovative insights into the future development of therapeutic agents for human CRC by offering novel chemical structure-activity relationship-related information.

Olverembatinib (HQP1351) Enhances Antitumor Effects of Chemotherapy in Philadelphia Chromosome-Positive (Ph⁺) Acute Lymphoblastic Leukemia (ALL)

Introduction Ph⁺ ALL is the most common subtype of B-cell- (B-) ALL in adults. BCR-ABL1 tyrosine kinase inhibitors (TKIs) are effective against chronic myeloid leukemia (CML) cells. However, treatment with TKIs fails to maintain disease remission in Ph⁺ B-ALL. Although the complete hematologic remission rate is very high, cytogenetic and molecular remission rates are low, so that TKIs are still considered as complements to chemotherapy and bridges to allogeneic stem cell transplantation. Olverembatinib is a novel, third-generation TKI with more potent activity against BCR-ABL1 and overcomes various resistant mutations, including the “gatekeeper” T315I, which confers resistance against all first- and second-generation TKIs. The aim of this study was to assess whether olverembatinib combined with chemotherapeutic agents vincristine or doxorubicin can promote anti-cancer effects in Ph⁺ ALL cells and explore the potential mechanism of action of the combinations. Methods Cell-based antiproliferation assays to evaluate the activities of olverembatinib alone or in combination with vincristine or doxorubicin were conducted using the CellTiter-Glo® Luminescent Cell Viability assay kits. Drug induced cell apoptosis was measured by flow cytometry. Potential mechanism of action analysis was conducted using western blot to evaluate the protein expression involved in BCR-ABL1 and apoptosis signaling pathways. Results Cell-based antiproliferation studies showed synergistic activity of olverembatinib in combination with vincristine or doxorubicin in Ph⁺ ALL SUP-B15 cells compared with single agents after 72 hours of treatment. The percentage of apoptotic cells induced by 24-hour treatment of olverembatinib (100 nM), vincristine (300 nM), and the combination were 27.7%, 27.2%, and 40.7%, respectively, while the percentage of apoptotic cells induced by 24-hour treatment of doxorubicin (100 nM) alone and in combination with olverembatinib (100 nM) were 35.3 and 74.1, respectively (apoptotic cells % in DMSO control group is 6.74). The combination significantly increased cell apoptosis compared with single agents ( p < 0.0001). Mechanistically, the combination of olverembatinib with vincristine showed synergistic effects in inhibiting protein expression and phosphorylation of BCR-ABL1, and downstream signal transducer and activator of transcription 5 (STAT5). The synergistic effects were also seen in decreased phosphorylation of AKT and ERK1/2. In addition, the combination significantly decreased protein levels of antiapoptotic MCL-1, BCL-2, and BCL-xL and increased protein levels of proapoptotic BAX and p53 upregulated modulator of apoptosis (PUMA) in a dose-dependent manner, which led to marked augmentation of cleavage of caspase-3 and poly (ADP-ribose) polymerase 1 (PARP). A similar pattern was also observed when olverembatinib was combined with doxorubicin. Conclusions Olverembatinib, a novel, third-generation TKI, in combination with chemotherapeutic agents vincristine or doxorubicin decreased MCL-1, BCL-2, and BCL-xL and increased BAX and PUMA in a dose-dependent manner, the combinations have demonstrated synergistic antitumor effects by enhancing apoptosis and antiproliferation in Ph⁺ ALL cells, which may provide an alternative approach for treatment of patients with Ph⁺ ALL.

Self-Assembly Driven Formation of Functional Ultralong "Artificial Fibers" to Mitigate the Neuronal Damage Associated with Alzheimer's Disease.

Fibrillation of amyloid beta (Aβ) is the key event in the amyloid neurotoxicity process that induces a chain of toxic events including oxidative stress, caspase activation, poly(ADP-ribose) polymerase cleavage, and mitochondrial dysfunction resulting in neuronal loss and memory decline manifesting as clinical dementia in humans. Herein, we report the development of a novel, biologically active supramolecular probe, INHQ, and achieve functional nanoarchitectures via a self-assembly process such that ultralong fibers are achieved spontaneously. With specifically decorated functional groups on INHQ such as imidazole, hydroxyquinoline, hydrophobic chain, and hydroxyquinoline molecules, these ultralong fibers coassembled efficiently with toxic Aβ oligomers and mitigated the amyloid-induced neurotoxicity by blocking the aforementioned biochemical events leading to neuronal damage in mice. These functional ultralong "Artificial Fibers" morphologically resemble the amyloid fibers and provide a higher surface area of interaction that improves its clearance ability against the Aβ aggregates. The efficacy of this novel INHQ molecule was ascertained by its high ability to interact with Aβ. Moreover, this injectable, ultralong INHQ functional "artificial fiber" translocates through the blood-brain barrier and successfully attenuates the amyloid-triggered neuronal damage and pyknosis in the cerebral cortex of wild-type mouse. Utilizing various spectroscopic techniques, morphology analysis, and in vitro, in silico, and in vivo studies, these ultralong INHQ fibers are proven to hold great promise for treating neurological disorders at all stages with a potential to replace the existing medications, reduce complications in the brain, and eradicate the amyloid-triggered neurotoxicity implicated in numerous disorders in human through a rare synergistic mechanism.

Effects of autophagy inhibitor 3-methyladenine on a diabetic mice model.

To investigate the role of autophagy inhibitor 3-methyladenine (3-MA) on a diabetic mice model (DM) and the potential mechanism. Male C57BL/6J mice were randomly divided into a normal control group (NC group) and an DM group. DM were induced by multiple low-dose intraperitoneal injection of streptozotocin (STZ) 60 mg/kg·d for 5 consecutive days. DM mice were randomly subdivided into untreated group (DM group), 3-MA (10 mg/kg·d by gavage) treated group (DM+3-MA group) and chloroquine (CQ; 50 mg/kg by intraperitoneal injection) treated group (DM+CQ group). The fasting blood glucose (FBG) levels were recorded every week. At the end of experiment, retinal samples were collected. The expression levels of pro-apoptotic proteins cleaved caspase-3, cleaved poly ADP-ribose polymerase 1 (PARP1) and Bax, anti-apoptotic protein Bcl-2, fibrosis-associated proteins Fibronectin and type 1 collagen α1 chain (COL1A1), vascular endothelial growth factor (VEGF), inflammatory factors interleukin (IL)-1β and tumor necrosis factor (TNF)-α, as well as autophagy related proteins LC3, Beclin-1 and P62 were determined by Western blotting. The oxidative stress indicators 8-hydroxydeoxyguanosine (8-OHdG) and malondialdehyde (MDA) were detected by commercial kits. Both 3-MA and CQ had short-term hypoglycemic effect on FBG and reduced the expression of VEGF and inflammatory factors IL-1β and TNF-α in DM mice. 3-MA also significantly alleviated oxidative stress indicators 8-OHdG and MDA, decreased the expression of fibrosis-related proteins Fibronectin and COL1A1, pro-apoptotic proteins cleaved caspase-3, cleaved PARP1, as well as the ratio of Bax/Bcl-2. CQ had no significant impact on the oxidative stress indicators, fibrosis, and apoptosis related proteins. The results of Western blotting for autophagy related proteins showed that the ratio of LC3 II/LC3 I and the expression of Beclin-1 in the retina of DM mice were decreased by 3-MA treatment, and the expression of P62 was further increased by CQ treatment. 3-MA has anti-apoptotic and anti-fibrotic effects on the retina of DM mice, and can attenuate retinal oxidative stress, VEGF expression and the production of inflammatory factors in the retina of DM mice. The underlying mechanism of the above effects of 3-MA may be related to its inhibition of early autophagy and hypoglycemic effect.

Fucoidan Enhances Cisplatin-induced Effects on SCC-25 Human Oral Cancer Cells by Inhibiting the PI3K/AKT Pathway.

Cisplatin is a drug for treating oral cancer. However, several previous studies indicate that oral cancer cells can develop resistance to cisplatin, which may result in a poor prognosis for patients with oral cancer. Fucoidan, a natural health product extracted from brown seaweed, has anticancer abilities against various types of cancer cell. This study evaluated whether fucoidan can enhance the sensitivity of oral cancer cells to cisplatin and explored the underlying mechanism. SCC-25 cells were used in the present study and treated with 0.3125 mg/ml fucoidan, 12.5 μg/ml cisplatin, or 0.3125 mg/ml fucoidan plus 12.5 μg/ml cisplatin for 48 h, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, enzyme-linked immunosorbent, and immunoblotting assays were performed to evaluate cell survival, cytokeratin-18 fragment release, and expression of markers of apoptosis and autophagy, respectively. Cotreatment with fucoidan enhanced cisplatin-induced reduction of SCC-25 cell survival compared to cisplatin alone. In addition, cotreatment also increased the expression of apoptosis markers, including activated caspase-8, activated caspase-9, activated caspase-3, and cleaved poly(ADP-ribose) polymerase (PARP), but did not increase the expression of the two autophagy markers studied, beclin and autophagy-related 12-autophagy-related 5 conjugate. Fucoidan significantly inhibited cisplatin-induced AKT serine/threonine kinase 1 activation, which promoted PARP cleavage, caspase-3 activation, and cytokeratin-18 fragment expression in SCC-25 cells. Fucoidan promoted cisplatin-induced effects by inhibiting phosphatidylinositol 4,5 bisphosphate 3 kinase/AKT serine/threonine kinase 1 activation induced by cisplatin. The results of this study may provide a basis for the possible application of the combination of fucoidan and cisplatin in the clinical treatment of oral cancer in the future to improve the prognosis of patients with oral cancer.

The BCL-2 family protein BCL-RAMBO interacts and cooperates with GRP75 to promote its apoptosis signaling pathway

The BCL-2 family protein BCL-RAMBO, also known as BCL2-like 13, anchors at the outer mitochondrial membrane and regulates apoptosis, mitochondrial fragmentation, and mitophagy. However, the mechanisms underlying the proapoptotic role of BCL-RAMBO remain unclear. In the present study, we demonstrated that BCL-RAMBO interacted with glucose-regulated protein 75 (GRP75), also known as heat shock protein family A member 9, and mortalin using co-immunoprecipitation and glutathione S-transferase-based pull-down assays. BCL-RAMBO interacted with GRP75 via its No BCL-2 homology domain. The interaction between BCL-RAMBO and GRP75 was confirmed by genetic interactions in Drosophila because a rough eye phenotype caused by the ectopic expression of BCL-RAMBO was partially suppressed by mutations in Hsc70-5, a mammalian GRP75 ortholog. In human embryonic kidney 293T cells, the co-expression of BCL-RAMBO and GRP75 facilitated an elevation in executioner caspase activity and poly (ADP-ribose) polymerase 1 (PARP-1) cleavage. In contrast, the knockdown of GRP75 suppressed elevated executioner caspase activity and PARP-1 cleavage in BCL-RAMBO-transfected cells. The mitochondrial release of cytochrome c induced by BCL-RAMBO was also attenuated by the knockdown of GRP75. These results indicate that GRP75 interacts with BCL-RAMBO and plays a crucial role in the BCL-RAMBO-dependent apoptosis signaling pathway.

Platinum(IV) Complexes as Inhibitors of STAT3 and Regulators of the Tumor Microenvironment To Control Breast Cancer.

Interplay between breast cancer (BC) cells and the tumor microenvironment (TME) influences the outcome of cancer treatment. Aberrant activation of signal transducer and activator of transcription 3 (STAT3) promotes the interaction and causes immunosuppression and drug resistance. Platinum(IV) complexes SPP and DPP bearing pterostilbene-derived axial ligand(s) were synthesized to inhibit the JAK2-STAT3 pathway in BC cells and regulate the TME. These complexes exerted remarkable antiproliferative activity against the triple-negative BC cells, suppressed the expression of phosphorylated STAT3 and STAT3-related cyclooxygenase-2 and IL-6, and activated caspase-3 and cleaved poly ADP-ribose polymerase, preventing the repair of DNA lesions and inducing apoptosis. Furthermore, DPP promoted the maturation and antigen presentation of dendritic cells, repressed the proliferation and differentiation of myeloid-derived suppressor cells and regulatory T cells, and facilitated the expansion of T cells. As a consequence, DPP showed excellent anticancer activity against BC with almost no general toxicity in vivo as a potential chemoimmunotherapeutic agent.

In vitro cytotoxic and apoptotic activity of the Mauritian marine sponge Neopetrosia exigua

Marine sponges belonging to the genus Neopetrosia represent a quasi-inexhaustible source of novel cytotoxic compounds. Yet studies delineating their molecular mechanisms of action in cancer cells remain scarce. We investigated the cytotoxic and apoptosis inducing potential of the Mauritian marine sponge Neopetrosia exigua derived crude extract, hexane and ethyl acetate fraction. Their cytotoxic activity was screened against four cancer cell lines and two non-malignant cell lines via the Alamar Blue metabolic assay. The level of intracellular reactive oxygen species (ROS) production, endogenous antioxidant enzyme activity (catalase and superoxide dismutase) and mitochondrial membrane potential were determined. The ability of the active extract to induce apoptosis in cancer cells and modulate the expression levels of apoptotic markers (caspases and polyADP-ribose polymerase (PARP)) was further evaluated via western blot. The ethyl acetate fraction (NEEAF) displayed the highest inhibitory effect with an IC50 of 6.87 μg/mL against the liver hepatocellular carcinoma cell line (HepG2). Mechanistically, NEEAF induced morphological hallmarks characteristic of apoptosis, increased ROS production, decreased catalase and superoxide dismutase activity and mitochondrial membrane depolarisation in a concentration-dependent manner compared to the control (p<0.05). In addition, NEEAF induced the activation of caspase-9, -7, -3 and cleavage of PARP. Overall, this study provides biochemical evidence for oxidative stress-mediated cytotoxicity and apoptosis in HepG2 cells by NEEAF. Further in-depth investigations are needed to isolate the active constituents, which may potentially lead to the development of novel anticancer therapeutics. Significance: Marine sponges represent an untapped goldmine of structurally unique compounds with interesting anticancer properties. This important initial investigative work will set the stage for more in-depth mechanistic studies and chemical characterisation of potentially novel bioactive compounds from the genus Neopetrosia. This work will also help to strengthen frameworks oriented towards the conservation of Neopetrosia species in the Western Indian Ocean region.

5,6-Dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) induces apoptosis in breast cancer cells through inhibiting of Mcl-1 expression

The effective treatment of breast cancer remains a profound clinical challenge, especially due to drug resistance and metastasis which unfortunately arise in many patients. The transcription inhibitor 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB), as a selective inhibitor of cyclin-dependent kinase 9, was shown to be effective in inducing apoptosis in various hematopoietic malignancies. However, the anticancer efficacy of DRB against breast cancer is still unclear. Herein, we demonstrated that administration of DRB to the breast cancer cell line led to the inhibition of cellular proliferation and induction of the typical signs of apoptotic cells, including the increases in Annexin V-positive cells, DNA fragmentation, and activation of caspase-7, caspase-9, and poly (ADP ribose) polymerase (PARP). Treatment of DRB resulted in a rapid decline in the myeloid cell leukemia 1 (Mcl-1) protein, whereas levels of other antiapoptotic proteins did not change. Overexpression of Mcl-1 decreased the DRB-induced PARP cleavage, whereas knockdown of Mcl-1 enhanced the effects of DRB on PARP activation, indicating that loss of Mcl-1 accounts for the DRB-mediated apoptosis in MCF-7 cells, but not in T-47D. Furthermore, we found that co-treatment of MCF-7 cells with an inhibitor of AKT (LY294002) or an inhibitor of the proteasome (MG-132) significantly augmented the DRB-induced apoptosis. These data suggested that DRB in combination with LY294002 or MG-132 may have a greater therapeutic potency against breast cancer cells.