[Corrigendum] (­)­Epigallocatechingallate induces apoptosis in B lymphoma cells via caspase­dependent pathway and Bcl­2 family protein modulation.

Subsequently to the publication of the above article, an interested reader drew to the authors' attention that, in Fig. 3 on p. 1510, the western blot images selected to portray the caspase 7 and PARP/cleaved PARP experiments were remarkably similar. After having referred to their original data, the authors realized that the PARP/cleaved PARP blots had been inadvertently duplicated in the figure. The revised version of Fig. 3, showing the correct data for the caspase­7 experiment, is shown below. The authors confirm that the errors made during the assembly of Fig. 3 did not adversely affect the major conclusions presented in this paper, and are grateful to the Editor of International Journal of Oncology for allowing them this opportunity to publish a corrigendum. They also apologize to the readership for any inconvenience caused. [International Journal of Oncology 46: 1507­1515, 2015; DOI: 10.3892/ijo.2015.2869].

Discussion of spinal cord neurons apoptosis and neuroprotection mechanism of NGF gene transfection mediated by recombinant adenovirus in EAE mice.

To investigate the spinal cord neuron apoptosis and neuroprotective mechanism of nerve growth factorganismsor (NGF) gene mediated by recombinant adenovirus (Ad-NGF) via peripheral transfection in mice with experimental autoimmune encephalomyelitis (EAE). Forty healthy female C57BL/6 mice were randomly divided into a control group, adenovirus (AdV) group, EAE group, and Ad-NGF transfection group; the control group received no treatment; the AdV group received adenovirus injection via the tail vein; the EAE and Ad-NGF transfection groups were induced with experimental autoimmune encephalomyelitis (EAE) using myelin oligodendrocyte glycoprotein 35-55 (MOG35-55), Ad-NGF transfection group received Ad-NGF injection via the tail vein, and daily neurological impairment scores were obtained. AQThe TUNEL method was employed to observe spinal neuron apoptosis in each group of mice; protein immunoblotting (western blot) and RT-PCR were used to measure NGF levels in the spinal cord tissues of each group, and western blotting was used to assess levels of cleaved caspase-3, Bax, and Bcl-2. ELISA and RT-PCR were employed to detect protein and mRNA levels of neuron-specific enolase (NSE) in spinal cord tissues, respectively. The control group and AdV mice did not develop symptoms. Compared to the EAE group, in the Ad-NGF transfection group, neurological function scores, TUNEL-positive cell counts, the ratio of NeuN + TUNEL to NeuN, levels of Bax and cleaved caspase-3 apoptotic proteins were significantly reduced, while Bcl-2 protein expression was increased. Expression levels of NGF, NGF-mRNA, NSE, and NSE-mRNA in spinal cord tissues were significantly elevated (P < 0.01). Immunofluorescence labeling revealed a significant punctate aggregation of apoptotic cells in spinal neurons of the EAE group, while the aggregation phenomenon was less pronounced in the Ad-NGF transfection group. Ad-NGF transfected by the periphery has a protective effect on spinal cord neurons in EAE mice by up-regulation NGF level, down-regulating apoptotic protein Caspase-3 in spinal cord neurons, inhibiting spinal cord neuron apoptosis and promoting NSE expression.

The protective effects of esculetin against Doxorubicin-Induced hepatotoxicity in rats: Insights into the modulation of Caspase, FOXOs, and heat shock protein pathways.

Doxorubicin (DOX) is an anthracycline antibiotic widely employed to treat carcinoma. Nevertheless, severe cardiotoxic side effects restrict its clinical use. Esculetin, a natural flavonoid, is found abundantly in plants. This study evaluated the protective effects of esculetin against DOX-induced hepatotoxicity in rat livers. Forty-eight rats were randomly divided into six groups with eight rats in each group: control (I), DOX (II), esculetin (III, 50 mg/kg), esculetin (IV, 100 mg/kg), DOX+esculetin 50 (V, DOX+esculetin 50 mg/kg), and DOX+esculetin 100 (VI, DOX+esculetin 100 mg/kg). The administration of esculetin effectively mitigated alterations in the measured biochemical parameters induced by DOX. Gene expression analyses demonstrated that esculetin treatment significantly reduced the DOX-induced expression of Foxo1, Hspa1a, Hsp4a, Hsp5a, Casp3, and Casp9 while increasing the DOX-induced expression of Foxo3. These findings suggest that esculetin, with its antioxidant and anti-inflammatory effects, might be a therapeutic option for protecting against DOX-induced hepatotoxicity.

ONC212, alone or in synergistic conjunction with Navitoclax (ABT-263), promotes cancer cell apoptosis via unconventional mitochondrial-independent caspase-3 activation.

Mitochondria-targeting agents, known as mitocans, are emerging as potent cancer therapeutics due to pronounced metabolic and apoptotic adaptations in the mitochondria of cancer cells. ONC212, an imipridone-family compound initially identified as a ClpP agonist, is currently under investigation as a potential mitocan with demonstrated preclinical efficacy against multiple malignancies. Despite this efficacy, the molecular mechanism underlying the cell death induced by ONC212 remains unclear. This study systematically investigates the mitochondrial involvement and signaling cascades associated with ONC212-induced cell death, utilizing HeLa and A549 cancer cells. Treated cancer cells exhibited characteristic apoptotic features, such as annexin-V positivity and caspase-3 activation; however, these occurred independently of typical mitochondrial events like membrane potential loss (ΔΨm) and cytochrome c release, as well as caspase-8 activation associated with the extrinsic pathway. Additionally, ONC212 treatment increased the expression of anti-apoptotic proteins Bcl-2 and Bcl-xL, which impeded apoptosis, as the overexpression of Bcl-2-GFP and Bcl-xL-GFP significantly reduced ONC212-mediated cell death. Furthermore, combining a sub-lethal dose of the Bcl-2/Bcl-xL inhibitor Navitoclax with ONC212 markedly augmented caspase-3 activation and cell death, still without any notable ΔΨm loss or cytochrome c release. Moreover, inhibition of caspase-9 activity unexpectedly augmented, rather than attenuated, caspase-3 activation and the subsequent cell death. Collectively, our research identifies ONC212 as an atypical mitochondrial-independent, yet Bcl-2/Bcl-xL-inhibitable, caspase-3-mediated apoptotic cell death inducer, highlighting its potential for combination therapies in tumors with defective mitochondrial apoptotic signaling.

Syk inhibitors reduce tau protein phosphorylation and oligomerization.

Spleen tyrosine kinase (Syk), a non-receptor-type tyrosine kinase, has a wide range of physiological functions. A possible role of Syk in Alzheimer's disease (AD) has been proposed. We evaluated the localization of Syk in the brains of patients with AD and control participants. Human neuroblastoma M1C cells harboring wild-type tau (4R0N) were used with the tetracycline off (TetOff) induction system. In this model of neuronal tauopathy, the effects of the Syk inhibitors-BAY 61-3606 and R406-on tau phosphorylation and oligomerization were explored using several phosphorylated tau-specific antibodies and an oligomeric tau antibody, and the effects of these Syk inhibitors on autophagy were examined using western blot analyses. Moreover, the effects of the Syk inhibitor R406 were evaluated in vivo using wild-type mice. In AD brains, Syk and phosphorylated tau colocalized in the cytosol. In M1C cells, Syk protein (72 kDa) was detected using western blot analysis. Syk inhibitors decreased the expression levels of several tau phosphoepitopes including PHF-1, CP13, AT180, and AT270. Syk inhibitors also decreased the levels of caspase-cleaved tau (TauC3), a pathological tau form. Syk inhibitors increased inactivated glycogen synthase kinase 3ß expression and decreased active p38 mitogen-activated protein kinase expression and demethylated protein phosphatase 2 A levels, indicating that Syk inhibitors inactivate tau kinases and activate tau phosphatases. Syk inhibitors also activated autophagy, as indicated by increased LC3II and decreased p62 levels. In vivo, the Syk inhibitor R406 decreased phosphorylated tau levels in wild-type mice. These findings suggest that Syk inhibitors offer novel therapeutic strategies for tauopathies, including AD.

FHOD3 shows clinical significance in progression of ovarian cancer through regulation of caspase-3 signaling pathway.

Ovarian cancer is a malignant disease threatening women's life. Traditional therapies bring little benefits for the patients with distant metastasis or recurrence. FHOD3 gene was reported to promote progression in cancer. However, the role of FHOD3 in ovarian cancer is not known yet. To investigate the role of FHOD3 gene in the progression of ovarian cancer and its molecular mechanism, FHOD3 gene was successfully knocked down in ovarian cancer cell lines. Then cell behaviors includes proliferation, migration, invasion, and apoptosis were detected. The data demonstrated that cell proliferation, migration, and invasion ability were suppressed after FHOD3 knockdown. Cell apoptosis was induced reversely. Moreover, caspase-3-mediated signaling pathway was activated after FHOD3 knockdown, and activity of caspase-3 further supported this finding. In addition, PARP inhibitor, Olaparib showed much more potent inhibition in ovarian cancer cells with FHOD3 knockdown. In clinical ovarian cancer tissues, FHOD3 gene showed increased expression compared to adjacent normal tissues. And FHOD3 gene expression level was negatively correlated to the patients' survival. Overall, these findings shed light on the significance of FHOD3 gene in progression of ovarian cancer. This study showed that FHOD3 gene might be exploited as a new target to improve the clinical outcome of ovarian cancer.

Beta-carotene ameliorates diabetic nephropathy in rats: involvement of AMPK/SIRT1/autophagy pathway.

OBJECTIVE: This study aimed to demonstrate the protective effect of beta-carotene against STZ-induced DN in rats and explore the possible underlying mechanisms that may have mediated such condition. MATERIAL AND METHODS: Wistar rats were allocated into four groups. Normal group received distilled water for 3 weeks. The other three groups were rendered diabetic by an intraperitoneal dose of STZ (50 mg/kg), 48 h later, group 2: received the vehicle and served as control, groups (3 &4) received orally beta-carotene in doses of 10 and 20 mg/kg, respectively for 3 weeks. Then serum and renal tissue were collected for biochemical, molecular, immunohistopathological, and histopathological examination. RESULTS: Beta-carotene ameliorated the reduction in body weight, reduced blood glucose, elevated serum insulin, reduced blood urea nitrogen, and serum creatinine levels. Beta-carotene elevated phosphorylated 5' adenosine monophosphate-activated protein kinase (p-AMPK)/AMPK, alleviated phosphorylated mammalian target of rapamycin (p-mTOR)/mTOR, reduced interleukin 1 beta (IL-1ß), increased Beclin 1, LC3II/LC3I, and reduced p62 renal contents. Moreover, it elevated renal SIRT1 gene expression and reduced renal tumor necrosis factor-alpha (TNF-α) and caspase-3 protein expressions. CONCLUSION: Beta-carotene exerted renoprotective effect against STZ-induced DN and histopathological alterations through alleviating hyperglycemia, attenuating inflammation, activating AMPK/SIRT1/autophagy pathway, and combating apoptosis.

Turkish coffee has an antitumor effect on breast cancer cells in vitro and in vivo.

BACKGROUND: Breast cancer is the most diagnosed cancer in women. Its pathogenesis includes several pathways in cancer proliferation, apoptosis, and metastasis. Some clinical data have indicated the association between coffee consumption and decreased cancer risk. However, little data is available on the effect of coffee on breast cancer cells in vitro and in vivo. METHODS: In our study, we assessed the effect of Turkish coffee and Fridamycin-H on different pathways in breast cancer, including apoptosis, proliferation, and oxidative stress. A human breast cancer cell line (MCF-7) was treated for 48 h with either coffee extract (5% or 10 v/v) or Fridamycin-H (10 ng/ml). Ehrlich solid tumors were induced in mice for in vivo modeling of breast cancer. Mice with Ehrlich solid tumors were treated orally with coffee extract in drinking water at a final concentration (v/v) of either 3%, 5%, or 10% daily for 21 days. Protein expression levels of Caspase-8 were determined in both in vitro and in vivo models using ELISA assay. Moreover, P-glycoprotein and peroxisome proliferator-activated receptor gamma (PPAR-γ) protein expression levels were analyzed in the in vitro model. ß-catenin protein expression was analyzed in tumor sections using immunohistochemical analysis. In addition, malondialdehyde (MDA) serum levels were analyzed using colorimetry. RESULTS: Both coffee extract and Fridamycin-H significantly increased Caspase-8, P-glycoprotein, and PPAR-γ protein levels in MCF-7 cells. Consistently, all doses of in vivo coffee treatment induced a significant increase in Caspase-8 and necrotic zones and a significant decrease in ß- catenin, MDA, tumor volume, tumor weight, and viable tumor cell density. CONCLUSION: These findings suggest that coffee extract and Fridamycin-H warrant further exploration as potential therapies for breast cancer.

CD39 inhibitor (POM-1) enhances radiosensitivity of esophageal squamous cell carcinoma (ESCC) cells by promoting apoptosis through the Bax/Bcl-2/Caspase 9/Caspase 3 pathway.

CD39 inhibitor (sodium polyoxotungstate, POM-1) has been reported to have antitumor effects. However, the synergistic effect of POM-1 with radiotherapy requires further elucidation. This study aimed to investigate the role and the molecular mechanism of POM-1 in esophageal squamous cell carcinoma (ESCC) radiosensitization. Firstly, the expression of CD39 in ESCC cells and normal esophageal epithelial cells were detected. Then radioresistant ESCC cells (Eca109R and KYSE150R) were constructed and CD39 expression was analyzed. Furthermore, the effect of POM-1 on radiosensitivity for parent cells and radioresistant cells were observed. Then, we analyzed the effect of POM-1 and CD39 siRNA on radiotherapy-induced apoptosis and determined whether POM-1 modulated the radioresistance of ESCC cells depending on the apoptotic signaling pathway. Finally, we validated the synergistic effect of POM-1 combined with radiotherapy in vivo. Our results showed that CD39 was highly expressed in ESCC cells and radioresistant ESCC cells (p < 0.05). POM-1 reduced radioresistance and proliferation of parent cells and radioresistant cells (p < 0.05). Further mechanistic exploration showed that inhibition of CD39 promoted radiation-induced apoptosis (p < 0.05). Bax knockdown reversed the effect of POM-1 on ESCC cells (p < 0.01). Animal experiments also validated that radiotherapy combined with POM-1 enhanced tumor inhibition in vivo (p < 0.05). These results suggested that POM-1 had synergistic effect with radiotherapy by enhancing cell apoptosis through Bax/Bcl-2 signal pathway in ESCC. The combination of POM-1 and radiotherapy is expected to enhance the anti-tumor effect in ESCC.

Exploring the Effects of S-Nitrosylation on Caspase-3 Modification and Myofibril Degradation of Beef In Vitro.

This study aimed to explore the effects of S-nitrosylation on caspase-3 modification and its subsequent effects on beef myofibril degradation in vitro. Recombinant caspase-3 was reacted with different concentrations of S-nitrosoglutathione (GSNO, nitric oxide donor) at 37 °C for 30 min and subsequently incubated with purified myofibrillar protein from bovine semimembranosus muscle. Results indicated that the activity of caspase-3 was significantly reduced after GSNO treatments (P < 0.05) and showed a dose-dependent inhibitory effect, which was attributed to the increased S-nitrosylation extent of caspase-3. LC-MS/MS analysis revealed that caspase-3 was S-nitrosylated at cysteine sites 116, 170, 184, 220, and 264. Moreover, the degradation of desmin and troponin-T was notably suppressed by S-nitrosylated caspase-3 (P < 0.05). To conclude, protein S-nitrosylation could modify the cysteine residues of caspase-3, which accounts for the reduced caspase-3 activity and further represses its proteolytic ability on beef myofibrillar protein.

AB045. E2F6 promotes temozolomide resistance in glioblastoma by enrichment in CARD6 and POSTN promoter region.

BACKGROUND: Glioblastoma (GBM) is the most aggressive primary malignant brain tumor. Temozolomide (TMZ) is the most used first-line chemotherapeutic agent for GBM after surgery, but acquired resistance to TMZ frequently leads to treatment failure and is a major challenge in the clinical treatment of GBM. Increasing evidence suggests that E2F transcription factor 6 (E2F6) is associated with a variety of tumor malignant biological behaviors and drug resistance, but its biological function and underlying molecular mechanisms in GBM are unknown. METHODS: The study investigated the levels of E2F6 in both TMZ-sensitive and TMZ-resistant GBM cells and tissues using Western blotting and immunofluorescence assays. In vitro experiments were conducted to explore the impact of E2F6 on TMZ resistance and glioma stem cell stemness. These experiments included Western blotting, colony formation assay, flow cytometry assay, and TdT-mediated dUTP nick-end labeling (TUNEL) assay. Bioinformatic analyses were conducted to investigate the mechanism behind the high expression of E2F6 in TMZ-resistant cells and its correlation with caspase recruitment domain 6 (CARD6) and disulfide-linked cell adhesion protein (POSTN). The study employed bioinformatic analyses, messenger RNA (mRNA) sequencing, chromatin immunoprecipitation sequencing assay, immunofluorescence, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blotting. To examine the function of E2F6, an intracranial xenograft tumor mouse model was used for in vivo experiments. RESULTS: It was found that CARD6 and POSTN were significantly associated with TMZ resistance and survival of GBM patients. E2F6 was up-regulated in TMZ-resistant cells and tissues. Knockdown of E2F6 down-regulated the expression of CARD6, promoted TMZ-induced apoptosis, and enhanced chemo-sensitivity, whereas its overexpression significantly increased TMZ resistance in vitro and in vivo. In addition, E2F6 can promote TMZ resistance through stem-like properties acquisition. We identified a signaling pathway related to E2F6 and POSTN, which maintains the self-renewal of GBM stem cells (GSCs). E2F6 concentrates in the promoter region of POSTN, thereby regulating the expression of GSCs-related genes cluster of differentiation 133 (CD133), Nestin, and sex-determining region Y-box 2 (SOX2), which may be involved in tumor metabolism and drug resistance processes. Down-regulation of E2F6 down-regulated the expression of POSTN and inhibited tumor growth in nude mice. CONCLUSIONS: These results suggest that the E2F6-CARD6/POSTN signaling axis regulates the malignant biological behaviors of GBM and TMZ resistance. These findings are expected to provide promising therapeutic targets for CARD6 overcoming GBM TMZ resistance.

Efficacy and mechanism of action of ginsenoside Rg3 on radiation proctitis in rats.

OBJECTIVE: Radiation proctitis (RP) refers to rectal injury caused by radiation treatment of pelvic and retroperitoneal malignancies, which has a major impact on the treatment prognosis and quality of life of patients with cancer. The tetracyclic triterpene saponin monomer ginsenoside Rg3 (GRg3), the primary bioactive ingredient in ginseng extracts, has therapeutic effects against RP in rats. Here, we validated its efficacy and elucidated its mechanism of action. METHODS: A rat RP model was established in 48 Wistar rats. Rats were randomly divided into control (untreated), irradiation, irradiation + dexamethasone, and irradiation + GRg3 (low-, medium-, and high-dose) groups. After 2 weeks' treatment, serum IL-4, IL-10, and TNF-α levels were tested by enzyme-linked immunosorbent assays. In rectal tissue, Ikbkb, Ikka, and Casp8 mRNA expression was detected by a reverse transcription-quantitative polymerase chain reaction. IKK-ß, IκB-α, p-IκB-α, p50, and caspase-8 protein levels were determined by western blot analysis. RESULTS: GRg3 significantly improved the general condition and histopathological damage in rats with RP. Moreover, GRg3 decreased the levels of factors that promote inflammation (TNF-α) and increased the levels of factors that reduce inflammation (IL-4 and IL-10). GRg3 markedly reduced the activation of NF-κB and caspase-8 signaling pathways. CONCLUSIONS: Thus, GRg3 may reduce the inflammatory response by blocking the NF-κB signaling pathway and improving the balance of inflammation-related factors. GRg3 may also inhibit intestinal cell apoptosis by suppressing the TNF-α/caspase-8 signaling cascade, thereby reducing radiological rectal injury. Our results verify that GRg3 is a promising therapeutic agent for RP treatment and shed light on its mechanism.

Eslicarbazepine induces apoptosis and cell cycle arrest in C6 glioma cells in vitro and suppresses tumor growth in an intracranial rat model.

BACKGROUND: Glioblastoma multiforme (GBM) is the most malignant brain tumor, with a poor prognosis and life expectancy of 14-16 months after diagnosis. The standard treatment for GBM consists of surgery, radiotherapy, and chemotherapy with temozolomide. Most patients become resistant to treatment after some time, and the tumor recurs. Therefore, there is a need for new drugs to manage GBM. Eslicarbazepine (ESL) is a well-known antiepileptic drug belonging to the dibenzazepine group with anticancer potentials. In this study, for the first time, we evaluated the potential effects of ESL on C6 cell growth, both in vitro and in vivo, and examined its molecular effects. METHODS: To determine the effect of ESL on the c6 cell line, cell viability, proliferation, and migration were evaluated by MTT assay, colony formation, and wound healing assay. Also, apoptosis and cell cycle were examined by flow cytometry, qRT-PCR, and western blotting. In addition, an intracranial model in Wistar rats was used to investigate the effect of ESL in vivo, and the tumor size was measured using both Caliper and MRI. RESULTS: The obtained results are extremely consistent and highly encouraging. C6 cell viability, proliferation, and migration were significantly suppressed in ESL-treated C6 cells (p < 0.001), as determined by cell-based assays. ESL treatment led to significant enhancement of apoptosis (p < 0.01), as determined by flow cytometry, and upregulation of genes involved in cell apoptosis, such as the Bax/Bcl2 ratio at RNA (p < 0.05) and protein levels (5.37-fold). Flow cytometric analysis of ESL-treated cells revealed G2/M phase cell cycle arrest. ESL-treated cells demonstrated 2.49-fold upregulation of p21 alongside, 0.22-fold downregulation of cyclin B1, and 0.34-fold downregulation of cyclin-dependent kinase-1 at the protein level. Administration of ESL (30 mg/kg) to male rats bearing C6 intracranial tumors also suppressed the tumor volume and weight (p < 0.01). CONCLUSIONS: Based on these novel findings, ESL has the potential for further experimental and clinical studies in glioblastoma.

Caspase 3 Expression Profiles in Meningioma Subtypes Based on Tissue Microarray Analysis.

Background/Aim: Concerning primary central nervous system neoplasms, meningiomas demonstrate the most common type in adults worldwide. Deregulation of apoptotic pathways in malignancies, including meningiomas, is correlated with chemoresistance and poor prognosis. Caspases represent crucial proteins that induce cell apoptosis. This study aimed to correlate caspase 3 protein expression levels to meningioma clinic-pathological features. Materials and Methods: A set of fifty (n=50) meningioma lesions was included in the current analysis including a broad spectrum of histopathological subtypes (meningotheliomatous, psammomatus, transitional, fibrous, angiomatous, microcystic, atypical and anaplastic). Immunohistochemistry was implemented on tissue microarray cores of selected paraffin blocks by applying an anti-caspase 3 antibody. Additionally, an image analysis protocol was also performed in the corresponding immunostained slides. Results: Caspase 3 protein over-expression was detected in 17/50 (34%) cases, whereas the remaining 33 cases (66%) were characterized by medium to low levels of the molecule. Caspase 3 expression was statistically significantly associated with the grade of the analyzed tumors and the mitotic index (p=0.002, p=0.001, respectively). Caspase 3 expression status was also correlated with the histotype of the selected meningiomas (p=0.016). Conclusion: Caspase 3 demonstrated low expression levels in a significant subset of the examined meningiomas correlated with differentiation grade, mitotic activity, and partially with specific histotypes. Agents that could enhance caspase 3 expression - inducing its apoptotic activity - represent a very promising area in oncology for developing novel treatment regimens.

Miltirone induces GSDME-dependent pyroptosis in colorectal cancer by activating caspase 3.

Colorectal cancer (CRC) is a common and malignant tumor, ranking as the third most common cancer in men and the second most common cancer in women. Pyroptosis, a recently described programmed cell death mechanism mediated by the GSDM family, has emerged as an immunogenic mechanism for chemotherapy drugs in tumor treatment. In this study, we discovered that Miltirone has the ability to reduce the viability of CRC cells (SW620 and HCT116) and cause the proteolytic cleavage of gasdermin E (GSDME) in CRC cells. It was also observed that inhibiting GSDME prevented pyroptotic cell death induced by Miltirone in SW620 and HCT116 cells. Furthermore, the main active component of Miltirone was found to effectively bind with caspase 3. SiRNA-mediated caspase 3 silencing and specific caspase 3 inhibitor Z-DEVD-FMK were shown to weaken Miltirone-induced GSDME-dependent cell death. The findings of the study suggest that Miltirone has the potential to inhibit the growth of CRC tumors in vivo by inducing pyroptotic cell death. This indicates that Miltirone could be a viable therapeutic agent for the treatment of CRC through GSDME-dependent pyroptosis. These results offer a promising new option for the clinical treatment of CRC.

Ibuprofen­derived nitric oxide donors with a high affinity to human serum albumin induce cell death in pancreatic cancer cells through a non­caspase 3/7­mediated pathway.

Nitric oxide (NO) has been reported to have a cytotoxic effect on various types of cancer. However, the efficient delivery of NO donors to tumors remains challenging. The present study used ibuprofen, which has a high binding affinity to human serum albumin (HSA). A total of two types of nitrated forms of ibuprofen, 4-[(nitrooxy)methyl]benzyl 2-(4-isobutylphenyl)propanoate [nitrated ibuprofen benzyl linker (NIB)] and 2-(nitrooxy)ethyl 2-(4-isobutylphenyl) propanoate [nitrated ibuprofen ethyl linker (NIE)], were synthesized. It was demonstrated that both NIB and NIE bound to the ibuprofen-binding site of HSA. Although NOx release was observed from NIB, but not NIE, intracellular NO release was detected from both NIB and NIE, which indicated that the mechanisms of NO release may be different for NIB and NIE. Both NIB and NIE induced concentration- and time-dependent cell death in human pancreatic cancer cells, whereas this cell death was not observed with ibuprofen, which could suggest that these cell death-inducing effects may be mediated by NO. The non-specific caspase inhibitor, z-VAD-FMK, inhibited cell death induced by NIB and NIE, but activation of caspase 3/7 was not observed. These results suggested that both NIB and NIE induced cell death through a non-caspase 3/7 pathway. The findings of the present study demonstrated that both NIB and NIE, as NO donors that could be retained in blood, may potentially be useful anti-cancer agent candidates in the future.

Multigenic family 110 (1 L-5-6 L) of African swine fever virus modulate cytokine genes expression in vitro.

BACKGROUND: African swine fever (ASF) is a viral disease that affects pigs and wild boars providing economic burden in swine industry. METHODS AND RESULTS: In this study, we investigated the effect of deleting the ASFV multigene family 110 (MGF110) fragment (1 L-5-6 L) on apoptosis modulation and the expression of proinflammatory cytokines. Gene expression in swine peripheral blood macrophages infected with either the parental "Volgograd/14c" strain or the gene-deleted "Volgograd/D(1L-5-6L) MGF110" strain was analyzed. Caspase-3 activity was 1.15 times higher in macrophages infected with the parental ASFV strain compared to the gene-deleted strain. Gene expression analysis of Caspase-3 (Cas-3), Interferon-A (IFN-A), Tumor Necrosis Factor A (TNF-A), B-cell Lymphoma-2 (Bcl-2), Nuclear Factor Kappa B (NF-kB), Interleukin-12 (IL-12), and Heat Shock Protein-70 (HSP-70) using RT-qPCR at various time points after infection revealed significant differences in expression profiles between the strains. The peak expression of cytokines (except NF-kB) occurred at 24 h post-infection with the "Volgograd/D(1L-5-6L) MGF110" strain. In samples infected with the ASFV "Volgograd/14c" strain, the most intense expression was observed at 72 and 96 h, except for Bcl-2 and NF-kB, which peaked at 6 h post-infection. The cytokine expression trend for the "Volgograd/D(1L-5-6L) MGF110" strain was more stable with higher expression values. CONCLUSION: The expression trend for the parental strain increased over time, reaching maximum values at 72 and 96 h post-infection, but the overall expression level was lower than that of the gene-deleted strain. These findings suggest that deleting the multigene family 110 members (1 L-5-6 L) contributes to ASFV attenuation without affecting virus replication kinetics.

Apoptotic endonuclease EndoG induces alternative splicing of Caspase-2.

Caspase-2 (Casp-2) is an enzyme that regulates the development of apoptosis upon alternative splicing of its mRNA. The long form of Casp-2 (Casp-2L) promotes apoptosis while the short form (Casp-2S) has decreased enzymatic activity and inhibits the development of apoptotic processes. However, very little is known about the mechanism of Casp-2 alternative splicing. Several endonucleases are known to participate in this process. The aim of this study was to determine the role of EndoG in regulation of Casp-2 alternative splicing. Strong correlation between expression levels of EndoG and Casp-2 splice-variants was found in CD4⁺ and CD8⁺ human T lymphocytes. Such correlation increased after incubation of these cells with etoposide. Increased expression of Casp-2S was determined during EndoG over-expression in CD4⁺ T-cells, after EndoG treatment of cell cytoplasm and nuclei and after nuclei incubation with EndoG digested cell RNA. Casp-2 alternative splicing was induced by a 60-mer RNA oligonucleotide in naked nuclei and in cells after transfection. The identified long non-coding RNA of 1016 nucleotides is the precursor of the 60-mer RNA oligonucleotide. Based on the results the following mechanism has been proposed. Casp-2 pre-mRNA is transcribed from the coding DNA strand while long non-coding RNA is transcribed from the template strand of the Casp-2 gene. EndoG digests long non-coding RNA and produces the 60-mer RNA oligonucleotide complementary to the Casp-2 pre-mRNA exon 9 and intron 9 junction place. Interaction of the 60-mer RNA oligonucleotide and Casp-2 pre-mRNA causes alternative splicing.

Extract of Araçá-Boi and Its Major Phenolic Compound, Trans-Cinnamic Acid, Reduce Viability and Inhibit Migration of Human Metastatic Melanoma Cells.

Cutaneous melanoma is an aggressive type of skin cancer that is recognized for its high metastatic potential and the challenges it presents in its treatment. There has been increasing interest in plant extracts and their potential applications in melanoma. The present study aimed to investigate the content of individual phenolic compounds in araçá-boi extract, evaluate their antioxidant activity, and explore their effects on cell viability, migration properties, oxidative stress levels, and protein expression in the human metastatic melanoma cell line SK-MEL-28. HPLC-DAD analysis identified 11 phenolic compounds in the araçá-boi extract. Trans-cinnamic acid was the main phenolic compound identified; therefore, it was used alone to verify its contribution to antitumor activities. SK-MEL-28 melanoma cells were treated for 24 h with different concentrations of araçá-boi extract and trans-cinnamic acid (200, 400, 600, 800, and 1600 µg/mL). Both the araçá-boi extract and trans-cinnamic acid reduced cell viability, cell migration, and oxidative stress in melanoma cells. Additionally, they modulate proteins involved in apoptosis and inflammation. These findings suggest the therapeutic potential of araçá-boi extract and its phenolic compounds in the context of melanoma, especially in strategies focused on preventing metastasis. Additional studies, such as the analysis of specific signaling pathways, would be valuable in confirming and expanding these observations.

Prognostic impact of caspase-8 mutation in oral cavity squamous cell carcinoma.

OBJECTIVE: Identifying the drive genes and inhibiting their significant signals were persistently the main concepts in cancer treatment. However, for oral cavity squamous cell carcinoma (OCSCC), the most influential genes for overall survival (OS) remain unclear. METHODS: A total of 120 OCSCC patients with corresponding pathologic specimens were collected in Taiwan. Whole-exome sequencing was done and the prognostic impact of each gene was analyzed. TCGA database was used to validate. RESULTS: The incidences of caspase-8 mutation were 22.1% and 10.9% in the Taiwan and TCGA cohort, respectively. In the Taiwan cohort, caspase-8 mutation was the most significant independent for OS with an adjusted hazard ratio (HR) ([95% CI]: 3.83 [1.84-7.99]). It was validated by the TCGA database (HR [95% CI]: 1.51 [1.00-2.29]). The 5-year OSs of the patients with or without caspase-8 mutation were 38.1% vs. 75.3% (p < 0.001) (HR [95% CI]: 3.264 [1.645-6.438]) in the Taiwan cohort, and 26.1% vs. 49.0% (p = 0.048) (1.513 [1.001-2.288]) in the TCGA cohorts, respectively. Caspase-8 mutation was also individually associated with poor prognosis for TNM stage I/II/III/IV, respectively. CASP8 R127* and R494*, defined as pathogenic mutations in ClinVar, were presented in both cohorts. CONCLUSIONS: Caspase-8 mutation was the most significant genetic alteration impacting prognosis.