PARP Cleavage Research Articles

Arginase Activity Inhibition With Thymoquinone Induces a Hybrid Type of Cell-Death in MDA-MB-231 Cell Line.

Arginase plays a crucial role in the urea cycle; it also has immunosuppressive and pro-tumor effects. The present study aimed to assess the effects of arginase inhibition by thymoquinone (2-Isopropyl-5-methyl-1,4-benzoquinone), an active compound of Nigella sativa, on cell death in the MDA-MB-231 triple-negative breast tumor cell line. Cell viability assays, Western blot analysis, and flow cytometry analysis were used to characterize oxidative stress and cell death. Our results showed that inhibition of arginase activity with thymoquinone significantly increased intracellular nitric oxide levels and resulted in overproduction of cellular and mitochondrial reactive oxygen species. Reductions in cell viability, cycle arrest, and increased cell death were also observed. Loss of transmembrane mitochondrial potential, activation of caspase-3, -7, and -9, cleavage of PARP, condensation and/or fragmentation of the nuclei, suggest that this cell death involved apoptosis. Furthermore, a cytoplasm vacuole formation and an increase in the ratio of [LC3-II/LC3-I] suggests a concomitant activation of autophagy with apoptosis. Altogether, the present study highlighted that arginase inhibition with thymoquinone induces a hybrid type of cell death defined as oxiapoptophagy. Thus, arginase inhibition with thymoquinone in the MDA-MB-231 cell line could be, in part, involved in the anticancer effect of thymoquinone.

Anti-cancer activity of synthetic gefitinib-1,2,3-triazole derivatives against Hela cells via induction of apoptosis

Cervical cancer ranks as the fourth most common cancer among women. However, the current treatments have significant side effects and limited therapeutic effects on advanced diseases, so it is necessary to discover better treatments for cervical cancer. The current study investigated the potential anticancer effects of a series of gefitinib-1,2,3-triazole derivative on Hela cells. Among the investigated, the target compound c13 showed good anticancer activity against Hela cells (IC50 = 5.66 ± 0.35 μM) compared with gefitinib (IC50 = 14.18 ± 3.19 μM). Moreover, compound c13 significantly inhibited the colony formation ability of Hela cells in a dose-dependent manner, accompanied by morphological changes in HeLa cells. Further investigations demonstrated that compound c13 triggered cell apoptosis and arrested the cell cycle at the G2/M phase in Hela cells. In addition, western blot analysis revealed that compound c13 upregulated the Bax/Bcl-2 ratio, and increased the levels of active caspase 3 and PARP1 cleavage, which suggested the involvement of the mitochondrial pathway in compound c13-induced apoptosis. In brief, these results indicated that compound c13 is a promising compound for the treatment of cervical cancer.

Coronin1A regulates the trafficking of alpha synuclein in microglia.

Microglia respond to cytotoxic protein aggregates associated with the progression of neurodegenerative disease. Pathological protein aggregates activate the microglial NLRP3 inflammasome resulting in proinflammatory signaling, secretion, and potentially pyroptotic cell death. We characterized mixed sex primary mouse microglia exposed to microbial stressors and alpha synuclein preformed fibrils (αsyn PFFs) to identify cellular mechanisms related to Parkinson's disease. Microglia package and release the endosome fate regulator Coronin1A (Coro1A) in EVs in an Nlrp3-dependent manner in widely used experimental activation conditions. We were surprised to find that Coro1A packaging and release was not Nlrp3-dependent in αsyn PFF exposure conditions. Coro1A-/- microglia exposed to αsyn PFFs trafficked more αsyn to lysosomal compartment increasing lysosomal membrane permeabilization. This corresponds to a decrease in αsyn released in EVs suggesting that Coro1A functions to shunt pathological proteins to a secretory pathway to attenuate lysosomal stress. αsyn PFF driven lysosomal stress resulting from Coro1a loss was associated with enhanced cytotoxicity. Intrinsic apoptosis signaling was unaffected, but we observed elevated cytosolic cathepsin B and the presence of a cathepsin associated 55kD PARP cleavage product. Post-mortem analysis of the PD mesencephalon supported a role for Coro1a in microglia, revealing elevated levels of Coro1A protein in human PD brains compared to those of healthy donors. Findings are relevant to the distribution of pathological αsyn and indicate that Coro1a protects microglia from lysosomal overload, inflammasome activation, and pyroptotic demise.Significance Statement Microglia are responsible for clearing toxic protein aggregates such as alpha synuclein (αsyn) in Parkinson's Disease (PD). PD is slowly progressive, implying that microglia are under proteinaceous stress for an extended time, maintaining some level of homeostasis while attempting to clear pathologically aggregated proteins. Pathological proteins can overload the lysosomes resulting in rupture, decreasing the ability of microglia to clear protein aggregates, and contributing to a hyperreactive inflammatory state. We determined that the protein Coronin1A functions in microglia to attenuate αsyn-induced lysosomal stress, preventing Nlrp3-inflammasome activation, and cell death. These findings identify a protective cellular mechanism operating in microglia that may contribute to the distribution of pathological proteins into the microenvironment.

WT161, a selective HDAC6 inhibitor, decreases growth, enhances chemosensitivity, promotes apoptosis, and suppresses motility of melanoma cells.

Histone deacetylase 6 (HDAC6) plays a critical role in tumorigenesis and tumor progression, contributing to proliferation, chemoresistance, and cell motility by regulating microtubule architecture. Despite its upregulation in melanoma tissues and cell lines, the specific biological roles of HDAC6 in melanoma are not well understood. This study aims to explore the functional effects and underlying mechanisms of WT161, a selective HDAC6 inhibitor, in melanoma cell lines. Cell proliferation was assessed using both 2D and 3D cell culture systems, including MTT assays, spheroid growth analyses, and colony formation assays. The interaction between WT161 and the chemotherapeutic agents temozolomide (TMZ) or dacarbazine (DTIC) was evaluated using the Chou-Talalay method. Apoptotic cell death was analyzed through flow cytometry, while migration, adhesion, and invasion assays were conducted to evaluate the motility capacities of melanoma cells. Western blot assays quantified α-tubulin acetylation (Lys40), PARP cleavage, and protein levels of β-catenin and E-cadherin. WT161 significantly reduced cell growth in both 2D and 3D cultures, decreased clonogenic capacity, and showed synergistic interactions with TMZ and DTIC. The inhibitor also induced apoptotic cell death and enhanced TMZ-induced apoptosis. Additionally, WT161 reduced cell migration and invasion while increasing cell adhesion. These effects were linked to changes in β-catenin and E-cadherin levels, depending on the specific cell type evaluated. Our study underscores the pivotal role of HDAC6 in melanoma progression, establishing it as a promising therapeutic target. We provide the first comprehensive evidence of WT161's anti-melanoma effects, setting the stage for further research into HDAC6 inhibitors as a potential strategy for melanoma treatment.

Peroxiredoxin 6 maintains mitochondrial homeostasis and promotes tumor progression through ROS/JNK/p38 MAPK signaling pathway in multiple myeloma

Peroxiredoxin 6 (PRDX6) is one of the Peroxiredoxin family members with only 1-Cys, using glutathione as the electron donor to reduce peroxides in cells. PRDX6 has been frequently studied and its expression was associated with poor prognosis in many tumors. However, the expression of PRDX6 in multiple myeloma (MM) and its relevance with MM remain unclear. In our study, we found that PRDX6 was overexpressed in MM patients. Its high expression was inversely correlated with prognosis but positively correlated with the levels of β2-microglobulin (B2M), lactate dehydrogenase (LDH), and International Staging System (ISS) stage of MM patients. Further, the deficiency of PRDX6 promoted MM cell lines (RPMI 8226, MM.1S, and U266) apoptosis significantly. Mechanically, PRDX6 serves as an anti-oxidative enzyme, and its deficiency led to over-accumulation of reactive oxygen species (ROS), resulting in oxidative stress, following the activation of MAPK signaling pathway, which manifested as phosphorylation of JNK and p38. Then, the expression of BAX and Bcl2 was imbalance, and the cascade cleavage of PARP and caspase 3 was increased, ultimately triggering cell apoptosis. In addition, oxidative stress decreased mitochondrial membrane potential (MMP), reduced gene expression levels of oxidative phosphorylation (OXPHOS), and increased in the density of mitochondrial crumpling, leading to mitochondrial structural abnormalities and dysfunction. Furthermore, PRDX6 deficiency combined with bortezomib induced a robust anti-tumor effect in MM cell lines. Finally, in vivo experiments also showed that the deficiency of PRDX6 inhibited tumor growth of tumor-bearing mice. Collectively, PRDX6 protects MM cells from oxidative damage and maintains mitochondrial homeostasis. And targeting PRDX6 is an attractive strategy to enhance the anti-tumor effect of bortezomib in MM.

Anti-Stemness and Anti-Proliferative Effects of Cadmium on Bovine Mammary Epithelial Cells.

Cadmium accumulation in the body can damage a variety of organs and impair their development and functions. In the present study, we investigated the effect of cadmium on the stemness and proliferation of normal bovine mammary epithelial cells (BMECs). Normal bovine mammary epithelial cells treated with cadmium chloride were assessed for the expression of stemness-related proteins and cell proliferation. Western blotting results found that exposure to different concentrations of cadmium (0, 1.25, 2.5, and 5 μm) for 48 h significantly increased Gli1 expression but unexpectedly decreased the expression of downstream stem cell-related proteins including BMI1, SOX2, and ALDH. However, we also observed that treatment with 5 μm cadmium for 48 h inhibited mammosphere formation using microscopy. In this study, cadmium exposure significantly reduced cell viability and mobility. Flow cytometry detection found that cadmium decreased the percentage of cells in the G0 phase but increased the percentage of cells in the S phase and the apoptosis rate. Furthermore, cadmium exposure significantly increased the levels of caspase-8, caspase-3, and PARP cleavage as assessed by western blotting. Our study uncovers a previously unrecognized role of cadmium in mammary cell stemness and suggests that cadmium may affect breast development by impairing normal stem cell self-renewal and inducing their apoptosis. Therefore, this study provides important scientific significance regarding whether heavy metal cadmium affects normal breast development.

Pentagalloyl Glucose from Bouea macrophylla Suppresses the Epithelial-Mesenchymal Transition and Synergizes the Doxorubicin-Induced Anticancer and Anti-Migration Effects in Triple-Negative Breast Cancer.

Background: Triple-negative breast cancer (TNBC) represents an aggressive form of breast cancer with few available therapeutic options. Chemotherapy, particularly with drugs like doxorubicin (DOX), remains the cornerstone of treatment for this challenging subtype. However, the clinical utility of DOX is hampered by adverse effects that escalate with higher doses and drug resistance, underscoring the need for alternative therapies. This study explored the efficacy of pentagalloyl glucose (PGG), a natural polyphenol derived from Bouea macrophylla, in enhancing DOX's anticancer effects and suppressing the epithelial-mesenchymal transition (EMT) in TNBC cells. Methods: This study employed diverse methodologies to assess the effects of PGG and DOX on TNBC cells. MDA-MB231 triple-negative breast cancer cells were used to evaluate cell viability, migration, invasion, apoptosis, mitochondrial membrane potential, and protein expression through techniques including MTT assays, wound healing assays, flow cytometry, Western blotting, and immunofluorescence. Results: Our findings demonstrate that PGG combined with DOX significantly inhibits TNBC cell proliferation, migration, and invasion. PGG enhances DOX-induced apoptosis by disrupting the mitochondrial membrane potential and activating caspase pathways; consequently, the activation of caspase-3 and the cleavage of PARP are increased. Additionally, the study shows that the combination treatment upregulates ERK signaling, further promoting apoptosis. Moreover, PGG reverses DOX-induced EMT by downregulating mesenchymal markers (vimentin and β-catenin) and upregulating epithelial markers (E-cadherin). Furthermore, it effectively inhibits STAT3 phosphorylation, associated with cell survival and migration. Conclusions: These results highlight the potential of PGG as an adjuvant therapy in TNBC treatment. PGG synergizes with DOX, which potentiates its anticancer effects while mitigating adverse reactions.

Apoptosis-Inducing and Proliferation-Inhibiting Effects of Doramectin on Mz-ChA-1 Human Cholangiocarcinoma Cells.

Cholangiocarcinoma is a malignant tumor that emerges in the intrahepatic or extrahepatic bile ducts. Doramectin (DOR), a third-generation derivative of avermectins (AVMs), is renowned for its low toxicity and high efficiency. However, no research has hitherto focused on the anti-cholangiocarcinoma effects of these drugs. In this study, we undertook a preliminary exploration of the mechanism through which DOR inhibits the viability of human cholangiocarcinoma cells (Mz-ChA-1) via transcriptome analysis and molecular validation at the cellular level. The results indicated that DOR could suppress the growth and proliferation of Mz-ChA-1 cells in a dose-dependent manner. Moreover, it significantly diminished their migration and invasion abilities. Cell cycle analysis disclosed arrest in the G1 phase, accompanied by an increase in p21 expression and a decrease in the levels of the cyclin E1 and CDK2 proteins. Additionally, DOR induced apoptosis via the ROS-triggered mitochondrial pathway. This was attested by an elevation in the BAX/BCL-2 ratio, the activation of caspase 3/7 and the cleavage of PARP1. These mechanistic insights underscore DOR's potential as a therapeutic agent against cholangiocarcinoma.

Cytotoxic effects of the standardized extract from Curcuma aromatica Salisb. rhizomes via induction of mitochondria-mediated caspase-dependent apoptotic pathway and p21-mediated G0/G1 cell cycle arrest on human gastric cancer AGS cells

ABSTRACT Curcuma aromatica Salisb. (C. aromatica) is one of the traditional herbs used to treat microbial infection, skin eruption, coronary heart disease, and other diseases, including cancer. However, the inhibitory effects and underlying mechanisms of action of C. aromatica on gastric cancer cells have not yet been fully elucidated. Our study aimed to examine the possible molecular mechanisms underlying the cytotoxic effects attributed to C. aromatica rhizome standardized extract against gastric cancer cells. The components of two major active compounds in C. aromatica rhizome extract were quantitatively analyzed using a simple and validated HPLC method. Cytotoxicity was determined in different gastric cancer and non-cancer cell lines. The biological activities of the extract targeting apoptosis and cell cycle-related genes on gastric cancer AGS cells were also investigated to elucidate the mechanisms relating to the anti-proliferative effect of C. aromatica rhizomes. The two major active compounds curdione and germacrone, in the C. aromatica extract were standardized to 0.64% and 1.12% w/w, respectively. The standardized extract (CAE) exerted cytotoxic effects on various cancer cells, whereas minimal effects at equivalent doses were noted for normal cells. CAE concentration-dependently suppressed growth of gastric cancer AGS cells via induction of apoptosis. Further studies revealed that CAE treatment disrupted mitochondrial membrane potential (ΔΨm), increased Bax/Bcl-2 ratio, and cytochrome c release, resulting in activation of caspase-9/-3 and subsequent cleavage of PARP. Further, the inhibitory effects of caspase-9/-3 expression by a synthetic pan-caspase inhibitor partially protected cells against apoptosis following CAE treatment. In addition, CAE significantly promoted cell death in AGS cells via an accumulation of cells in the G0/G1 phase. This effect was associated with upregulation of the CDK inhibitor p21 and downregulation of cyclin D1, cyclin E, CDK4, and CDK2 expression. Our data indicated that CAE exerted anti-proliferative activity by activating the mitochondria-mediated caspase-dependent apoptotic pathway and arresting the p21-mediated G0/G1 cell cycle on human gastric cancer AGS cells.

RNA-binding protein HuR inhibition induces multiple programmed cell death in breast and prostate cancer

The RNA-binding protein Hu antigen R (HuR) plays a pivotal role in cancer progression, and previous studies have demonstrated its involvement in suppressing cell death in cancer. However, the precise mechanisms underlying HuR inhibition-induced cell death remain elusive. Here, we investigated the impacts of HuR functional inhibition via the small molecule inhibitor KH-3 on cell proliferation, colony formation, and cell death across multiple cancer cell lines, with an emphasis on breast and prostate cancers. KH-3 treatment induced apoptotic cell death of various cancer cell lines, as well as autophagy-associated cell death and ferroptosis. Remarkably, KH-3-induced cell death was partially rescued by an autophagy inhibitor and a ferroptosis inhibitor. The anti-tumor effects of KH-3 were further validated in two mouse xenograft models of human prostate cancer. Mechanistically, KH-3 reduced the expression of HuR targets involved in apoptosis and ferroptosis suppression, including cFLIP and SLC7A11, respectively. Moreover, cFLIP silencing enhanced Caspase-8 activation as well as PARP cleavage in both breast cancer and prostate cancer cells. Both KH-3-induced pharmacological HuR inhibition and RNA interference-mediated HuR knockdown reduced the expression of SLC7A11. Additionally, KH-3 also reduced XIAP and Survivin, enhancing the activation of multiple caspases and leading to apoptosis. This study highlights the critical roles of HuR in programmed cell death regulation, advocating HuR inhibition as a promising anti-tumor strategy for cell-death-inducing cancer therapy.

Targeting Non-Apoptotic Pathways with the Cell Permeable TAT-Conjugated NOTCH1 RAM Fragment for Leukemia and Lymphoma Cells.

Targeting nonapoptotic cell death offers a promising strategy for overcoming apoptosis resistance in cancer. In this study, we developed Tat-Ram13, a 25-mer peptide that fuses the NOTCH1 intracellular domain fragment RAM13 with a cell-penetrating HIV-1 TAT, for the treatment of T-cell acute lymphoblastic leukemia with aberrant NOTCH1 mutation. Tat-Ram13 significantly downregulated NOTCH1-target genes in T-ALL cell lines. Furthermore, the peptide had potent cytotoxic effects on various human leukemia and lymphoma cell lines. However, it did not affect normal lymphocytes and monocytes, some subsets of leukemia cells, or adherent tumor cells. This cell-selective cytotoxic activity was closely correlated with the peptide uptake via macropinocytosis in leukemia cells. In leukemia cells, Tat-Ram13 triggered rapid cell death. This cell death involved mitochondrial membrane depolarization and extracellular release of lactate dehydrogenase and high-mobility group box-1 protein without activation of caspase-3 or cleavage of PARP-1. These results suggest that Tat-Ram13 cell death is nonapoptotic and mediated by rapid plasma membrane rupture. Moreover, alanine scanning analysis identified four critical hydrophobic amino acids in the RAM13 domain essential for its cytotoxicity. Consequently, these results suggest that Tat-Ram13 is a tumor-selective, nonapoptotic cell death-inducing agent for treating refractory leukemia and lymphomas with apoptosis resistance.

Chloroquine sensitises hypoxic colorectal cancer cells to ROS-mediated cell death via structural disruption of pyruvate dehydrogenase kinase 1.

Chloroquine (CQ), an autophagy antagonist, has been recently explored as a repurposable medicine for cancer; however the exact mechanism of its action is still not known. The present study investigated the effect of CQ on colorectal cancer cells to elucidate the underlying molecular mechanisms. We report for the first time that CQ suppresses hypoxia-induced growth and survival of HCT-116 cells by reducing glycolytic capacity and NAD+ production through inhibition of PDK1. Furthermore, in silico and in vitro studies revealed that CQ induces structural alteration in the PDK1 protein, leading to its destabilization and promotes its enhanced degradation by proteases. This degradation is in turn inhibited by the MG-132 protease inhibitor. Moreover, CQ-induced suppression of PDK1 results in mitochondrial damage through excessive production of ROS, as reflected by the reduction in mitochondrial membrane potential, which in turn triggers apoptosis through PARP cleavage and Caspase activation. These findings advocate CQ as a promising repurposable chemotherapeutic for colorectal cancer and a novel inhibitor of PDK1.

Nuclear IMPDH2 controls the DNA damage response by modulating PARP1 activity

Nuclear metabolism and DNA damage response are intertwined processes, but the precise molecular links remain elusive. Here, we explore this crosstalk using triple-negative breast cancer (TNBC) as a model, a subtype often prone to DNA damage accumulation. We show that the de novo purine synthesis enzyme IMPDH2 is enriched on chromatin in TNBC compared to other subtypes. IMPDH2 chromatin localization is DNA damage dependent, and IMPDH2 repression leads to DNA damage accumulation. On chromatin, IMPDH2 interacts with and modulates PARP1 activity by controlling the nuclear availability of NAD+ to fine-tune the DNA damage response. However, when IMPDH2 is restricted to the nucleus, it depletes nuclear NAD+, leading to PARP1 cleavage and cell death. Our study identifies a non-canonical nuclear role for IMPDH2, acting as a convergence point of nuclear metabolism and DNA damage response.

EXTH-62. FOCAL ADHESION KINASE (FAK) IN CHROMOSOME 8 GAIN-ASSOCIATED MALIGNANT PERIPHERAL NERVE SHEATH TUMORS (MPNST)

Abstract OBJECTIVES Malignant peripheral nerve sheath tumors (MPNST) are aggressive sarcomas often associated with Neurofibromatosis type 1 (NF1), a genetic syndrome driven by overactivation of the RAS pathway. MEK inhibitors (MEKi) target downstream components of the RAS pathway and have been approved for NF1 patients with plexiform neurofibromas (PN). However, MPNST often develop resistance to MEKi. Our lab recently discovered the critical role of chromosome 8 (Chr8) genes in MPNST. The Chr8 gene, protein tyrosine kinase 2 (PTK2), also known as focal adhesion kinase (FAK) is overexpressed in multiple cancers, suggesting its importance in tumor progression. In this study, we aim to evaluate FAK as a therapeutic target for MPNST and investigate the FAKi and RAF/MEKi combination. METHODS A CRISPR gRNA screen was used to identify Chr8 genes involved in MPNST survival. The effect of FAK shRNA knockdown and a FAK inhibitor (FAKi), alone or combined with a MEKi, in MPNST cells was assessed using IncuCyte proliferation assays and multiple murine models. RESULTS The CRISPR gRNA screen identified 57 genes on Chr8 important for MPNST cell survival, including PTK2/FAK. Knockdown of FAK by shRNA in MPNST inhibited cell proliferation in vitro and reduced tumorigenesis in vivo. Consistently, the FAKi defactinib significantly inhibited the proliferation of MPNST (IC50-values: 1.72-4.65μM). Moreover, the defactinib/avutometinib combination was synergistic in vitro and the combination of FAKi with RAF/MEKi reduced tumor growth more that either drug alone in Chr8-gain MPNST PDX models. Avutometinib induced compensatory activation of FAK, while defactinib/avutometinib combination decreased phosphorylation of FAK, ERK1/2 and STAT3, and augmented cleavage of Caspase-3 and PARP-1, shown using the WES protein simple system. CONCLUSION These results demonstrate the potential therapeutic efficacy of FAK inhibitors, both alone and in combination with RAF/MEKi, for the treatment of MPNST.

Effect of Soy Isoflavone on Prostate Cancer Cell Apoptosis Through Inhibition of STAT3, ERK, and AKT.

Genistein, an isoflavone found in soybeans, exhibits antioxidant, anti-inflammatory, and anticancer properties. This study explored the molecular mechanisms behind genistein's anticancer effects in prostate cancer DU145 cells. In this study, genistein decreased cell viability, increased annexin V-PE(+) cells, and enhanced the sub-G0/G1 peak by flow cytometric analysis. Increased reactive oxygen species increased mitochondrial depolarization indicating mitochondrial dysfunction and inhibition of ATP formation were also observed in genistein-treated DU145 cells. Genistein upregulated p53 at the mRNA and protein levels and increased caspase-3/7 activity along with the cleavage of Bax, procaspase-3, and PARP. With the increasing genistein concentrations, the percentage of cells in the sub-G0/G1 peak and G2/M phase increased, which was inhibited by treatment with the pan-caspase inhibitor Z-VAD together with 100 μM genistein, which had little toxicity to normal prostate epithelial HPrEC cells. Genistein treatment simultaneously inhibited the activation of STAT3 and other closely related oncogenic kinases such as AKT and ERK and p38 and decreased VEGF expression. Taken together, these results suggest that genistein inhibits the growth of DU145 cells and induces apoptosis by inhibiting STAT3, AKT, ERK, and p38 which provides a molecular basis for the anticancer activity of genistein and suggests its potential as a valuable therapeutic candidate for prostate cancer.

Synergistic effect of the sphingosine kinase inhibitor safingol in combination with 2'-nitroflavone in breast cancer.

Sphingosine kinase-1 (SPHK1), the enzyme that catalyzes the synthesis of the pro-oncogenic molecule sphingosine-1-phosphate, is commonly upregulated in breast cancer cells and has been linked with poor prognosis and progression by promoting cell transformation, proliferation, angiogenesis, and metastasis. Therefore, SPHK1-targeting drugs have been proposed for breast cancer treatment, with better antitumor results when they are combined with chemotherapy. Previously, we demonstrated that the synthetic flavonoid 2'-nitroflavone (2'NF) exerted a potent and selective antiproliferative effect in murine HER2-positive LM3 mammary tumor cells. As we found that these cells overexpress SPHK1, we decided to explore the antitumor action of the combination of SPHK inhibitors (safingol or SKI-II) with 2'NF. In vitro assays showed that the combination induced a synergistic antiproliferative effect in LM3 cells. Similar results were obtained when human HER2-positive MDA-MB-453 breast cancer cells were treated with the combination of 2'NF/safingol. We also found that safingol potentiated the 2'NF apoptotic effect in both cell lines. The synergistic antitumor effect was confirmed in vivo in an LM3 syngeneic breast cancer model. Moreover, western blot analysis of tumor lysates revealed that combined treatment increased PARP cleavage and Bax protein levels and decreased anti-apoptotic Bcl-xL and Bcl-2 protein levels. Additionally, mice treated with both compounds showed no histopathological effects on different organ tissues. In summary, these findings suggest that the combination safingol/2'NF can be proposed as a potential therapeutic strategy for HER2-positive breast cancer treatment. KEY MESSAGES: The combination safingol/2'-nitroflavone exerts a synergic antitumor action in vitro. Safingol potentiates 2'-nitroflavone apoptotic effect in breast cancer cells. Safingol enhances the 2'-nitroflavone antitumor activity in vivo in breast cancer.

Photochemical Metal-Free synthesis and biological Assessment of isocryptolepine analogues targeting estrogen receptor Alpha in breast cancer cells

The aim of this study was to develop a new series of isocryptolepines and evaluate their antiproliferative and antiestrogenic activities on cancer cells. A series of isocryptolepine derivatives were synthesized using developed one-pot photochemical, metal-free protocol, employing readily available 2-arylindoles as starting compounds. The resulting isocryptolepines demonstrated (sub)micromolar inhibitory activity against selected breast cancer cell lines. The IC50 values ​​of lead compound 3c against hormone-dependent breast cancer types (MCF7 and T47D) were 0.3 μM and 0.12 μM, respectively, and significantly greater than 3 μM against estrogen receptor α (ERα)-deficient cell lines, MDA-MB-231 and HCC1954, respectively. To assess the antiestrogenic potency of compound 3c, MCF7 cells were transfected with a plasmid containing a luciferase reporter gene under the control of an estrogen-responsive element (ERE), creating the MCF7/ERE-LUC cell subline. In these cells, luciferase activity was induced by the natural ERα ligand, 17β-estradiol (E2). Compound 3c inhibited luciferase activity by 50 % at a concentration of 0.12 μM, highlighting its potent inhibitory effect on ERα. Molecular modeling further indicated that compound 3c could directly bind to ERα.Compound 3c induced apoptosis, as evidenced by PARP cleavage and downregulation of p-Bcl-2 and Bcl-2, and demonstrated synergistic effects in combination with the chemotherapeutic agent 5-fluorouracil. Compound 3c also showed selectivity towards hormone-dependent breast cancer cells, likely targeting ERα − a key driver in this cancer subtype. In summary, we report the development of a first-in-class antiestrogenic isocryptolepine with notable pro-apoptotic efficacy.

A Novel Combinatorial Regimen Using Sorafenib and Uttroside B, A US FDA-designated 'Orphan Drug', for the Treatment of Hepatocellular Carcinoma.

Sorafenib (Sor) is the first-line treatment option in clinics for treating advanced unresectable hepatocellular carcinoma (HCC). However, acquired chemoresistance and adverse side effects associated with Sor monotherapy limit its clinical benefits. We have previously reported the exceptional anti-HCC potential of uttroside B (Utt-B), a furostanol saponin isolated in our lab from Solanum nigrum Linn. leaves. The current study has evaluated the supremacy of a combinatorial regimen of Sor and Utt-B over Sor monotherapy. MTT assay was used for In vitro cytotoxicity studies. A clonogenic assay was conducted to assess the anti-proliferative effect of the combination. Annexin V/PI staining, confocal microscopy, FACS cell cycle analysis, and Western blotting experiments were performed to validate the pro-apoptotic potential of the combination in HepG2 and Huh7 cell lines. Pharmacological safety evaluation was performed in Swiss albino mice. Our results indicate that Utt-B augments Sor-induced cytotoxicity in HepG2 and Huh7 cells. The combination inhibits the proliferation of liver cancer cells by inducing apoptosis through activation of the caspases 7 and 3, leading to PARP cleavage. Furthermore, the combination does not induce any acute toxicity in vivo, even at a dose five times that of the effective therapeutic dose. Our results highlight the potential of Utt-B as an effective chemosensitizer, which can augment the efficacy of Sor against HCC and circumvent Sor-induced toxic side effects. Moreover, this is the first and only report to date on the chemosensitizing potential of Utt-B and the only report that demonstrates the therapeutic efficacy and pharmacological safety of a novel combinatorial regimen involving Utt-B and Sor for combating HCC.

Manuka Essential Oil Triggers Apoptosis and Activation of c-Jun N-Terminal Kinase in Fibroblasts and Fibrosarcoma Cells.

Manuka essential oil has long been used in traditional medicine, though the effects of the oil on cancer cells have limited studies. The goal of this project was to treat cancer cell lines with manuka essential oil at different concentrations and to ascertain the effects on the cell proliferation of normal fibroblast (CUA-4) and on fibrosarcoma (HT-1080) cells. Cell lines were grown on 24-well plates, and subconfluent cultures were treated with varying concentrations of manuka oil for 24 h. The effect of the oil on proliferation and viability was measured through direct cell counting using trypan blue dye exclusion and through the use of an MTT assay. As the concentration of oil increased, proliferation of all cell lines tested decreased with increasing dosage, concurrently with a decrease in MTT activity. To determine if the decrease in cell numbers observed from manuka oil treatment is the result of apoptosis, PARP cleavage assays were performed, confirming that the treatment caused apoptosis in both normal fibroblasts and fibrosarcoma cells. The stress-activated MAPK protein, JNK, was activated by manuka essential oil treatment, occurring synergistically with a decrease in MKP-1 expression.

Fostamatinib Inhibits the Proliferation of Ovarian Cancer Cells Through Apoptosis Induction.

Ovarian cancer remains a significant challenge due to its high mortality rate and poor prognosis, especially in advanced stages. Despite treatment advancements, issues with resistance and recurrence persist, highlighting the urgent need for new and effective therapies. This study aimed to evaluate fostamatinib, an oral spleen tyrosine kinase inhibitor initially developed for autoimmune diseases, as a potential treatment for ovarian cancer. The effects of fostamatinib on ovarian cancer cell lines were assessed using WST-1 assays for cell proliferation. Apoptosis was evaluated through TUNEL assays, DNA fragmentation analysis, and flow cytometry. Western blot analysis was used to detect cleavage of apoptotic proteins, including caspase-3 and PARP, and flow cytometry analyzed cell cycle changes. Fostamatinib treatment resulted in a dose- and time-dependent reduction in ovarian cancer cell growth and induced apoptosis, as indicated by increased TUNEL-positive cells, DNA fragmentation, and rises in both early and late apoptosis. Western blot analysis showed increased cleavage of apoptotic proteins, including caspase-3 and PARP. Flow cytometry also demonstrated an increase in the sub-G1 phase of the cell cycle, further supporting apoptosis induction. Fostamatinib, by inhibiting cell proliferation and inducing apoptosis, shows promise as a repurposed therapeutic agent for ovarian cancer, potentially offering a new approach to improve patient outcomes.