Inhibition of JNK-Mediated Autophagy Promotes Proscillaridin A- Induced Apoptosis via ROS Generation, Intracellular Ca+2 Oscillation and Inhibiting STAT3 Signaling in Breast Cancer Cells.

Breast cancer is the most heterogenous cancer type among women across the world. Despite concerted efforts, breast cancer management is still unsatisfactory. Interplay between apoptosis and autophagy is an imperative factor in categorizing therapeutics for cancer treatment. Proscillaridin A (PSD-A), a well-known cardiac glycoside used for cardiac arrest and arrythmias, has been unveiled in many cancer types but the underlying mechanism for apoptosis and autophagy in breast cancer is not fully understood. In our study, PSD-A restricted cell growth, inhibited STAT3 activation and induced apoptosis and autophagy in breast cancer cells via ROS generation and Ca+2 oscillation. Pretreatment of NAC and BAPTA-AM restored PSD-A induced cellular events in breast cancer cells. PSD-A induced apoptosis via DNA fragmentation, caspase-cascade activation, PARP cleavage, mitochondrial dysfunction, Bax/Bcl-2 proteins modulation and ER chaperone GRP78 inhibition along with decreased phosphorylation of ERK1/2. Inhibition of STAT3 activation was found to be associated with decreased phosphorylation of SRC. Moreover, PSD-A induced events of autophagy i.e. conversion of LC3-I to LC3-II, and Atg3 expression via JNK activation and decreased mTOR and AKT phosphorylation. In this study, pretreatment of SP600125, a JNK inhibitor, reduced autophagy and enhanced STAT3 inhibition and apoptosis. Additionally, SB203580, a commercial p38 inhibitor, stimulated STAT3 activation and improved autophagic events rate in breast cancer cells, displaying the role of the MAPK signaling pathway in interplay between apoptosis and autophagy. Our data suggest that the rate of apoptotic cell death is improved by blocking JNK-induced autophagy in PSD-A treated MCF-7 and MDA-MB-231 breast cancer cells.

miR-421 up-regulation by the oleanolic acid derivative K73-03 regulates epigenetically SPINK1 transcription in pancreatic cancer cells leading to metabolic changes and enhanced apoptosis.

SPINK1 overexpression promotes cancer cell aggressiveness and confers chemo-resistance to multiple drugs in pancreatic cancer. Oleanolic acid (OA) derivatives possess active effects against different cancers. Here we report the effect of K73-03, a new novel OA derivative, against pancreatic cancer through mitochondrial dysfunction via miR-421/SPINK1 regulation. We examined the binding ability of miR-421 with SPINK1-3'UTR Luciferase reporter assays. Moreover, miR-421/SPINK1 expressions in pancreatic cancer, with or without K73-03 treatment, were evaluated. Cells viability, migration, autophagy, mitochondrial function and apoptosis were examined with or without K73-03 treatment. We established that the K73-03 effect on the miR-421 that plays a crucial role in the regulation of SPINK1 in pancreatic cancer. Our findings indicated that K73-03 inhibited the mitochondrial function that led to inducing autophagy and apoptosis through epigenetic SPINK1 down-regulation via miR-421 up-regulation in pancreatic cancer. Furthermore, the inhibition of miR-421 expression in pancreatic cancer cells abolished the efficacy of K73-03 against SPINK1 oncogenic properties. We found an interesting finding that the interaction between miR-421 and SPINK1 is related to mitochondrial function through the effect of K73-03. Further, SPINK1 appear to be the molecular targets of K73-03 especially more than gemcitabine.

MicroRNA-4316 inhibits gastric cancer proliferation and migration via directly targeting VEGF-A.

BACKGROUND AND AIMS: microRNAs (miRNAs) have been reported to regulate proliferation and migration by down-regulating the expression of target genes. The aims of this study were to investigate whether miR-4316 inhibited proliferation and migration by downregulating vascular endothelial growth factor A (VEGF-A) and its clinical significance in gastric cancer (GC). METHODS: The clinical tissues of the GC patients for miR-4316 and VEGF-A were detected by qRT-PCR. The protein levels of VEGF-A and c-Met were determined by western blotting. Cell Proliferation, migration, and colony forming assays were conducted to show whether miR-4316 affects proliferation by CCK-8, migration by transwell, wound healing and colony formation assays. The bioinformatic methods and luciferase reporter assay were applied to detect the relationship between miRNA and VEGF-A on its targeting 3-untranslated regions (3-UTRs). CCK-8, colony formation, wound healing, and transwell assay were performed to explore the function of miR-4316. RESULTS: The results of qRT-PCR indicated that miR-4316 expression level was significantly downregulated in human GC tissues and GC cell lines compared with their control. miR-4316 inhibited proliferation, migration and colony formation in GC cell lines by reducing VEGF-A. And western blot results indicated that miR-4316 significantly inhibited GC through repressing VEGF-A and c-Met. The investigation of Luciferase assay indicated that VEGF-A is a direct target gene of miR-4316. CONCLUSIONS: miR-4316 suppressed proliferation and migration of GC through the VEGF-A gene. MiR-4316 acts as a tumor suppressor by targeting VEGF-A and this indicated that MiR-4316 might be a potential therapeutic target for GC.

Brevilin A Inhibits STAT3 Signaling and Induces ROS-Dependent Apoptosis, Mitochondrial Stress and Endoplasmic Reticulum Stress in MCF-7 Breast Cancer Cells.

PURPOSE: Breast cancer is the most common malignancy among women across the globe. Despite concerted efforts to improve the prevailing treatment modalities, the overall prognosis of breast cancer remains unsatisfactory. Recently, antiproliferative activity of Brevilin A (Brv-A), a sesquiterpene lactone compound of Centipeda minima, has been unveiled in various cancer types. Here, we have explored anticancer activity of Brv-A in MCF-7 breast carcinoma cells by targeting various pathways. MATERIALS AND METHODS: Cell proliferation rate was determined by CCK-8 and clonogenic assay. Cellular morphological changes were observed under phase contrast microscope while calcein-AM and PI was used for live/dead assay. Cell cycle assay was performed by flow cytometry. Apoptotic cell percentage was determined by Hoechst 33258 staining and flow cytometric analysis. ROS generation and mitochondrial membrane potential were measured using commercially available kits while protein expression was measured by Western blotting. RESULTS: In our study, Brv-A exerted antiproliferative effect through mitotic arrest at G2/M phase of cell cycle and induced apoptosis in MCF-7 cells in a dose-dependent manner. Induction of apoptosis by Brv-A was found to be associated with ROS generation by targeting NOX2 and NOX3, mitochondrial dysfunction (MMP dissipation and Bcl-2 family proteins modulation), DNA fragmentation, JNK and p38 MAPK activation, endoplasmic reticulum (ER) stress by increasing Bip/GRP78, ATF4 and CHOP protein expressions and inhibition of STAT3 activation via decreased phosphorylation of JAK2 and SRC. Pretreatment of NAC, a ROS scavenger, partially reversed the aforesaid cellular events indicating ROS generation as the primary event to modulate cellular targets for induction of apoptosis. Besides, Brv-A has also been documented for inhibition of cell migration via decrease in COX-2 and MMP-2 expression. CONCLUSION: Taken together, Brv-A induces G2/M phase arrest, ROS-dependent apoptosis, ER stress, mitochondrial dysfunction and inhibits STAT3 activation in MCF-7 cells signifying it to be one of the potential anticancer therapeutics in future.

Molecular Mechanisms of Anticancer Activities of Puerarin.

Medicinal plants are a vital source of natural products (NPs) that can cure cancer through modulation of different pathways, including oxidative stress, extrinsic and intrinsic apoptosis, cell cycle, inflammation, NF-kB, PI3K/AKT/mTOR, AMPK (JNK), MEK/ERK (Raf)-MEK-ERK and autophagy. Puerarin (Pue), an important NP belonging to the isoflavone glycoside group, is derived from Pueraria lobata (Willd.) Ohwi, Pueraria thomsonii Benth, and Pueraria tuberosa (Willd.). This NP was approved by the Chinese Ministry of Health for the treatment of different diseases in 1993, but it was also later reported to exhibit anticancer activity. Pue causes cancer cells death through modulation of different mechanisms including oxidative stress, intrinsic and extrinsic, Survivin and XIAP, PI3K/AKT/mTOR, Ras-Raf-MEK-ERK, JNK, cell cycle, AMPK, NF-kB, inflammation and autophagy pathways. Therefore, this review compiles for the first time the studies about the anticancer mechanism of Pue and provides comprehensive information about the anticancer effects of Pue. This review may serve as a basis for future research and clinical treatment.

Phosphocreatine Improves Cardiac Dysfunction by Normalizing Mitochondrial Respiratory Function through JAK2/STAT3 Signaling Pathway In Vivo and In Vitro.

Diabetic cardiomyopathy (DCM) is one of the common cardiovascular complications in patients with diabetes. Accumulating evidence has demonstrated that DCM is thoroughly related to mitochondrial energy impairment and increases the generation of reactive oxygen species (ROS). Therefore, an ongoing study is developing strategies to protect cardiac mitochondria from diabetic complications, especially from hyperglycemia. Phosphocreatine (PCr) plays a major metabolic role in cardiac muscular cells including intracellular concentration of ATP which affects the activity of the myocardium. We hypothesized that PCr might improve oxidative phosphorylation and electron transport capacity in mitochondria impaired by hyperglycemia in vivo and in vitro. Also, we aimed to evaluate the protective effect of PCr against DCM through the JAK2/STAT3 signaling pathway. The mitochondrial respiratory capacity from rats and H9C2 cells was measured by high-resolution respirometry (HRR). Expressions of proteins Bax, Bcl-2, caspase 3, caspase 9, cleaved caspase 3, and cleaved caspase 9, as well as JAK2/STAT3 signaling pathways, were determined by western blotting. ROS generation and mitochondrial membrane potential (MMP) were measured with fluorescent probes. Type 1 diabetes mellitus was induced in Wistar male rats by a single intraperitoneal injection of streptozotocin (STZ) (80 mg/kg body weight). Our results revealed that PCr possessed protective effects against DCM injury by improving the mitochondrial bioenergetics and by positively exerting protective effects against DCM in vivo and in vitro, not only improving diabetes symptom, resulting in changes of cardiac tissue using hematoxylin and eosin (H&E) stain, but also ameliorating biochemical changes. Moreover, PCr increased Bcl-2, caspase 3, and caspase 9 protein expressions and decreased Bax, cleaved caspase 3, and cleaved caspase 9 expressions as well as the JAK2/STAT3 signaling pathway. In conclusion, PCr improves mitochondrial functions and exerts an antiapoptotic effect in vivo and in vitro exposed to oxidative stress by hyperglycemia through the JAK2/STAT3 signaling pathway. Our findings suggest that PCr medication is a possible therapeutic strategy for cardioprotection.

GPX2 suppression of H2O2 stress regulates cervical cancer metastasis and apoptosis via activation of the ß-catenin-WNT pathway.

BACKGROUND: Increasing evidence suggests that glutathione peroxidase 2 (GPX2) plays important roles in the tumorigenesis and progression of various human cancers, such as colorectal carcinomas and lung adenocarcinomas. However, the role of GPX2 in cervical cancer is unclear. In this study, we identified the role of GPX2 in cervical cancer tissues and cell lines. MATERIALS AND METHODS: The basal mRNA and protein expression of GPX2 in cervical cancer cells and a series of key molecules in the epithelial to mesenchymal transition (EMT) and WNT/ß-catenin pathways were examined via real time fluorescence quantitative PCR (qRT-PCR) and Western blot assays. The biological phenotype of the cervical cancer cell lines was detected by the cloning formation and transwell assays, and intracellular reactive oxygen species (ROS) levels were detected by flow cytometry. Finally, the GPX2 expression level in 100 clinical cervical tissues was examined by immunohistochemistry. RESULTS: We found that GPX2 was highly expressed in cervical cancer tissues compared to normal individuals and promoted the proliferation and metastasis of cervical cancer cells, and this promotion correlated with the activation of EMT and WNT/ß-catenin signaling in vitro. GPX2 was determined to reduce apoptotic damage by reducing hydroperoxides. According to the characteristics and verification of GPX2, this series of phenotypes is clearly related to oxidative stress in cells. Furthermore, we verified that GPX2 was highly expressed in cervical cancer tissues and promoted the metastasis of cervical cancer. CONCLUSION: In summary, we found that GPX2 was highly expressed in cervical cancer cells and promoted the proliferation and metastasis of cervical cancer by affecting oxidative stress. Our study provides a new target for the clinical treatment of cervical cancer.