Berberine increases the expression of cytokines and proteins linked to apoptosis in human melanoma cells.

BACKGROUND: Melanoma is the most lethal form of skin cancer, and its incidence has increased considerably in the last decades. Melanoma presents difficult treatment with strong resistance of tumor cells, due to its extremely invasive nature with high capacity to metastases. Berberine (BBR), an isoquinoline alkaloid, is a molecule found in several medicinal plants, and has been studied in several diseases, demonstrating antimicrobial, antidiabetic and anti-inflammatory properties and anti-tumorigenic effects. METHODS AND RESULTS: In SK-MEL-28 cells, 50 µM BBR treatment for 24 h decreased cell viability by 50 percent. This concentration generated cell death both by early apoptosis and necrosis, with an increase in the DNA damage index. BBR increased (*p < 0.05) the proportion of cells in G1/G0 phase and decreased (###p < 0.005) the percentage of cells in S phase. The alcaloid increased (****p < 0.001) ROS production compared to untreated controls with an increase in activated caspase 3 and phosphorylated p53 protein levels. In addition, BBR significantly enhanced ERK as well as both pro- and anti-inflammatory cytokine expression compared to untreated controls. CONCLUSIONS: BBR has important antiproliferative effects and may be alone or in adjunct therapy a promising candidate for melanoma treatment, a cancer with great incidence and high lethality.

Activated mitochondrial apoptosis in hESCs after dissociation involving the PKA/p-p53/Bax signaling pathway.

Human embryonic stem cells (hESCs) are highly fragile with massive cell death after dissociation into single cells, which seriously hampers their applications. The mechanism underlying the massive cell death after dissociation still remains elusive. Here, the expression of apoptosis-related proteins, cell survival and mitochondrial membrane potential in dissociated hESCs before and after the treatments with a protein kinase A (PKA) inhibitor H89 and p53 inhibitor Pifithrin α were investigated, respectively. Protein interactions were identified by immunoprecipitation and immunofluorescence. The results show that the dissociation causes Caspase-dependent apoptosis in hESCs mediated by mitochondrial pathway with the up-regulation of pro-apoptotic proteins, decrease in mitochondrial membrane potential and elevation in pro-apoptotic Cyto c release, which are obviously suppresses by H89. The dissociation-induced increase of phosphorylated p53 Ser15 (p-p53) is suppressed by Pifithrin α which also rescues the elevated levels of pro-apoptotic proteins in mitochondrial pathway. During the dissociation-induced apoptosis, PKA/p-p53/Bax signaling pathway is identified by immunoprecipitation and immunofluorescence showing the most likely interaction between them. These results indicate that dissociation induces mitochondrial apoptosis in hESCs involving PKA/p-p53/Bax signaling pathway, which not only give new insights into the apoptosis mechanism of dissociated hESCs, but also provide clues for developing potential strategies to promote hESC survival after dissociation.

Changes in the expression level of IL-17A and p53-fibrinolytic system in smokers with or without COPD.

COPD is a chronic airway inflammatory disease characterized mainly by neutrophil airway infiltrations. The neutrophil airway inflammation is mainly mediated through a key player like the pro-inflammatory cytokine IL-17A which is involved in the modulation of p53-fibrinolytic system. This study was undertaken to examine the molecular changes for the expressions of IL-17A and p53-fibrinolytic system in smokers with or without COPD. Blood and serum samples were collected from ten patients of smokers having COPD and ten samples from smokers without COPD and ten healthy control subjects. Western blot analyses were performed to evaluate the expressions of IL-17A, p53 and PAI-1. Apoptosis was assessed by immunoblot for cleaved caspase-3. In addition, FEV% was also determined of these patients. qRT-PCR was done to detect the gene expression study from the blood samples on p53-fibrinolytic components. A significant difference was found in the expression levels of IL-17A in smokers with COPD patient when compared to smokers without COPD and the control subjects. Similarly the smokers with COPD showed significant increase in the fibrinolytic component PAI-1 as well as in expression levels of p53 when compared to smokers without COPD and normal subjects. Increased cleaved caspase-3 may also promote apoptosis.The expression pattern of the IL-17A in chronic obstructive pulmonary distress syndrome samples was increased as compared of those of normal samples, and their main role in the regulation of and p53-fibrinolytic system makes these components as a predictive prominent component in smokers with COPD.

Hypoxia triggers cardiomyocyte apoptosis via regulating the m6A methylation-mediated LncMIAT/miR-708-5p/p53 axis.

Long-time hypoxia induced cardiomyocyte apoptosis is an important mechanism of myocardial ischemia (MI) injury. Interestingly, long noncoding RNA myocardial infarction-associated transcript (LncMIAT) has been involved in the regulation of MI injury; however, the underlying mechanism by which LncMIAT affects the progression of hypoxia-induced cardiomyocyte apoptosis remains unclear. In the present study, hypoxia was found to promote cardiomyocyte apoptosis through an increased expression of LncMIAT in vitro. Biological investigations and dual-luciferase gene reporter assay further revealed that LncMIAT was able to bind with miR-708-5p to upregulate the p53-mediated cell death of the cardiomyocytes. Silencing of LncMIAT or overexpression of miR-708-5p led to a significant reduction in p53-mediated cardiomyocyte apoptosis. The methylated RNA immunoprecipitation (MeRIP)-qPCR results showed that hypoxia exerted its effects on LncMIAT through AKLBH5-N6-methyladenosine (m6A) methylation and therefore hypoxia was shown to trigger HL-1 cardiomyocyte apoptosis via the m6A methylation-mediated LncMIAT/miR-708-5p/p53 axis. Silencing of AKLBH5 significantly alleviated the m6A methylation-mediated LncMIAT upregulation and p53-mediated cardiomyocyte apoptosis, while promoted miR-708-5p expression. Taken together, the present study highlighted that LncMIAT could act as a key biological target during hypoxia-induced cardiomyocyte apoptosis. In addition, it was shown that hypoxia could promote cardiomyocyte apoptosis through regulation of the m6A methylation-mediated LncMIAT/miR-708-5p/p53 signaling axis.

Nuclear HKII-P-p53 (Ser15) Interaction is a Prognostic Biomarker for Chemoresponsiveness and Glycolytic Regulation in Epithelial Ovarian Cancer.

In epithelial ovarian cancer (EOC), carboplatin/cisplatin-induced chemoresistance is a major hurdle to successful treatment. Aerobic glycolysis is a common characteristic of cancer. However, the role of glycolytic metabolism in chemoresistance and its impact on clinical outcomes in EOC are not clear. Here, we show a functional interaction between the key glycolytic enzyme hexokinase II (HKII) and activated P-p53 (Ser15) in the regulation of bioenergetics and chemosensitivity. Using translational approaches with proximity ligation assessment in cancer cells and human EOC tumor sections, we showed that nuclear HKII-P-p53 (Ser15) interaction is increased after chemotherapy, and functions as a determinant of chemoresponsiveness as a prognostic biomarker. We also demonstrated that p53 is required for the intracellular nuclear HKII trafficking in the control of glycolysis in EOC, associated with chemosensitivity. Mechanistically, cisplatin-induced P-p53 (Ser15) recruits HKII and apoptosis-inducing factor (AIF) in chemosensitive EOC cells, enabling their translocation from the mitochondria to the nucleus, eliciting AIF-induced apoptosis. Conversely, in p53-defective chemoresistant EOC cells, HKII and AIF are strongly bound in the mitochondria and, therefore, apoptosis is suppressed. Collectively, our findings implicate nuclear HKII-P-p53(Ser15) interaction in chemosensitivity and could provide an effective clinical strategy as a promising biomarker during platinum-based therapy.

Luteoloside Protects the Uterus from Staphylococcus aureus-Induced Inflammation, Apoptosis, and Injury.

Luteoloside is a flavonoid extracted from several natural herbs that exhibits anti-microbial and anti-inflammation properties. Our study mainly identified the anti-inflammatory mechanism of action of luteoloside in Staphylococcus aureus-induced endometritis. Histopathological observations and myeloperoxidase (MPO) activity showed that luteoloside could protect the uterus from S. aureus-induced damage and ameliorate the infiltration of inflammatory cells. Quantitative PCR (qPCR) and ELISA analysis also revealed that luteoloside could decrease the expression of the pro-inflammatory cytokines TNF-α, IL-1ß, and IL-6 and increase the expression of the anti-inflammatory cytokine IL-10 both in vivo and in vitro. However, western blot analysis revealed that luteoloside inhibited the expression of TLR2 and IL-8 and inhibited the phosphorylation of IκBα and NF-κB p65. Moreover, luteoloside inhibited the apoptosis of endometrial epithelial cells (EECs), suppressed the phosphorylation of p53, and decreased the expression of caspase-3 induced by S. aureus. Furthermore, this study showed that luteoloside inhibited the expression of Bax but increased the expression of Bcl-2. These results indicate that luteoloside has anti-inflammatory properties by inhibiting the TLR2 and NF-κB signaling pathways and plays an anti-apoptotic role in S. aureus-induced endometritis, which may be valuable for the clinical treatment of S. aureus-induced inflammation.

Benzo[a]pyrene-induced cell cycle progression occurs via ERK-induced Chk1 pathway activation in human lung cancer cells.

Benzo[a]pyrene (B[a]P) is a potent lung carcinogen derived from tobacco smoking and environmental contamination. This study aimed to investigate the signal transduction pathway responsible for B[a]P-induced non-small cell lung cancer (NSCLC) development. We exposed the human NSCLC cell lines Calu-1, CL3, H1299, CH27, H23, and H1355 to B[a]P and assessed cell cycle progression using flow cytometry. Expression of cell cycle mediators was measured using Western blot analyses and electrophoretic mobility shift assays (EMSAs). B[a]P exposure dramatically induced S-phase accumulation in H1355 cells. Phospho-p53 (Ser15 and Ser20), phospho-ERK, phospho-p38, and Bax were significantly increased in H1355 cells whereas phospho-Rb was decreased in these cells. In addition, B[a]P induced phosphorylation of checkpoint kinase-1 (Chk1) but not Chk2. EMSA experiments revealed a slower migrating band after c-Myc bound the E-box in response to B[a]P treatment, which was abolished upon the addition of the ERK inhibitor PD98059 in H1355 cells. Phospho-ERK inhibition and dominant negative mutant Chk1 expression reversed B[a]P-induced S phase accumulation and downregulated phospho-Chk1 and phospho-ERK expression. Taken together, these results suggest that activation of ERK and its downstream mediator Chk1 may contribute to B[a]P-induced S phase accumulation in H1355 cells. The results could help in the development of lung cancer treatments that target the Chk1 pathway through ERK.

Improved ataxia telangiectasia mutated kinase inhibitor KU60019 provides a promising treatment strategy for non-invasive breast cancer.

It has previously been reported that KU60019, as a highly effective radiosensitizer, inhibits the DNA damage response and blocks radiation-induced phosphorylation of key ataxia telangiectasia mutated targets in human glioma cells. The present study investigated whether KU60019 affects cell physiological activities and strengthens the efficacy of doxorubicin-induced DNA damage. It was demonstrated that the compound suppressed the proliferation of MCF-7 cells and significantly increased chemosensitization. In addition, KU60019 (without doxorubicin) inhibited MCF-7 cell motility and invasion, potentially by acting on the phosphorylated-Akt and E-cadherin signaling pathways. Although the majority of MCF-7 cells were arrested at the G1/S phase following treatment with KU60019, the combination of the two compounds did not result in such a marked effect on the cell cycle. In conclusion, KU60019 is a potent chemosensitizer in combination with doxorubicin, therefore, it may provide a promising strategy for non-invasive breast cancer.

Resveratrol contributes to chemosensitivity of malignant mesothelioma cells with activation of p53.

Resveratrol is a naturally occurring polyphenolic phytoalexin with chemopreventive properties. We previously reported a synergistic anti-proliferative effect of resveratrol and clofarabine against malignant mesothelioma (MM) cells. Here, we further investigated molecular mechanisms involved in the synergistic interaction of these compounds in MM MSTO-211H cells. Resveratrol, in combination with clofarabine, time-dependently induced a strong cytotoxic effect with the nuclear accumulation of phospho-p53 (p-p53) in MSTO-211H cells, but not in normal mesothelial MeT-5A cells. Combination treatment up-regulated the levels of p-p53, cleaved caspase-3, and cleaved PARP proteins. Gene silencing with p53-targeting siRNA attenuated the sensitivity of cells to the combined treatment of two compounds. Analyses of p53 DNA binding assay, p53 reporter gene assay, and RTP-CR toward p53-regulated genes, including Bax, PUMA, Noxa and p21, demonstrated that induced p-p53 is transcriptionally active. These results were further confirmed by the siRNA-mediated knockdown of p53 gene. Combination treatment significantly caused the accumulation of cells at G1 phase with the increases in the sub-G0/G1 peak, DNA ladder, nuclear fragmentation, and caspase-3/7 activity. Taken together, these results demonstrate that resveratrol and clofarabine synergistically elicit apoptotic signal via a p53-dependent pathway, and provide a scientific rationale for clinical evaluation of resveratrol as a promising chemopotentiator in MM.

Ursolic acid induces apoptosis of SW480 cells via p53 activation.

The purpose of this study is to assess the effect of ursolic acid, a triterpene, on induction of apoptosis in human colon adenocarcinoma cells, SW480. The cells treated with nontoxic concentration of ursolic acid showed the presence of apoptotic bodies and DNA fragmentation in a dose dependent manner. Both the expression level and transcriptional activity of p53, an apoptotic protein, were remarkably up-regulated in cells treated with ursolic acid compared to blank group. In addition, the expression levels of p-p53, acetyl-p53, NF-κB, p-p21, Bax, Caspase-3 and Caspase-9, apoptotic proteins, were increased in cells treated with ursolic acid compared to blank group. Above results demonstrate that ursolic acid induces the activation of p53, NF-κB and Bax, leading to the enhancement of p21 transcriptional activity and activation of Caspase-9 and -3, thus finally induces apoptosis of SW480 cells.