A Novel Approach to Unraveling the Apoptotic Potential of Rutin (Bioflavonoid) via Targeting Jab1 in Cervical Cancer Cells

Rutin has been well recognized for possessing numerous pharmacological and biological activities in several human cancer cells. This research has addressed the inhibitory potential of rutin against the Jab1 oncogene in SiHa cancer cells, which is known to inactivate various tumor suppressor proteins including p53 and p27. Further, the inhibitory efficacy of rutin via Jab1 expression modulation in cervical cancer has not been yet elucidated. Hence, we hypothesized that rutin could exhibit strong inhibitory efficacy against Jab1 and, thereby, induce significant growth arrest in SiHa cancer cells in a dose-dependent manner. In our study, the cytotoxic efficacy of rutin on the proliferation of a cervical cancer cell line (SiHa) was exhibited using MTT and LDH assays. The correlation between rutin and Jab1 mRNA expression was assessed by RT-PCR analysis and the associated events (a mechanism) with this downregulation were then explored via performing ROS assay, DAPI analysis, and expression analysis of apoptosis-associated signaling molecules such as Bax, Bcl-2, and Caspase-3 and -9 using qRT-PCR analysis. Results exhibit that rutin produces anticancer effects via inducing modulation in the expression of oncogenes as well as tumor suppressor genes. Further apoptosis induction, caspase activation, and ROS generation in rutin-treated SiHa cancer cells explain the cascade of events associated with Jab1 downregulation in SiHa cancer cells. Additionally, apoptosis induction was further confirmed by the FITC-Annexin V/PI double staining method. Altogether, our research supports the feasibility of developing rutin as one of the potent drug candidates in cervical cancer management via targeting one such crucial oncogene associated with cervical cancer progression.     

Fisetin Inhibits UVB‐induced Cutaneous Inflammation and Activation of PI3K/AKT/NFκB Signaling Pathways in SKH‐1 Hairless Mice

Solar ultraviolet B (UVB) radiation has been shown to induce inflammation, DNA damage, p53 mutations and alterations in signaling pathways eventually leading to skin cancer. In this study, we investigated whether fisetin reduces inflammatory responses and modulates PI3K/AKT/NFκB cell survival signaling pathways in UVB‐exposed SKH‐1 hairless mouse skin. Mice were exposed to 180 mJ cm^?2 of UVB radiation on alternate days for a total of seven exposures, and fisetin (250 and 500 nmol) was applied topically after 15 min of each UVB exposure. Fisetin treatment to UVB‐exposed mice resulted in decreased hyperplasia and reduced infiltration of inflammatory cells. Fisetin treatment also reduced inflammatory mediators such as COX‐2, PGE₂ as well as its receptors (EP1–EP4) and MPO activity. Furthermore, fisetin reduced the level of inflammatory cytokines TNFα, IL‐1β and IL‐6 in UVB‐exposed skin. Fisetin treatment also reduced cell proliferation markers as well as DNA damage as evidenced by increased expression of p53 and p21 proteins. Further studies revealed that fisetin inhibited UVB‐induced expression of PI3K, phosphorylation of AKT and activation of the NFκB signaling pathway in mouse skin. Overall, these data suggest that fisetin may be useful against UVB‐induced cutaneous inflammation and DNA damage.     

Piperine loaded zinc oxide nanocomposite inhibits the PI3K/AKT/mTOR signaling pathway via attenuating the development of gastric carcinoma: In vitro and in vivo studies

Gastric cancer (GC) is the fifth most cancer type and the third most cause of cancer-associated deaths worldwide along with the 5-year survival rate is less the 30%. This investigation was aimed to synthesis the piperine-loaded zinc oxide nanocomposite (ZnO-Pip-NC) and investigating its anticancer activity against the GC by in vitro and in vivo models by the inhibiting the apoptotic and PI3K/Akt/mTOR signaling pathways. The synthesized ZnO-Pip-NC was characterized by different techniques. The cytotoxicity of zinc oxide, piperine and the formulated ZnO-Pip-NC was tested against the AGS cells by MTT assay. The intracellular ROS level, mitochondrial membrane potential, and apoptotic cell necrosis in the AGS cells was examined by fluorescent staining techniques. The expression of apoptotic and PI3K/Akt/mTOR signaling markers were inspected by western blotting and the expression of pro0inflammatory markers analyzed by RT-PCR technique. The antioxidant levels were examined by standard methods and histopathology of gastric mucosa was analyzed. The ZnO-Pip-NC treatment appreciably inhibited the AGS cell viability. ZnO-Pip-NC treated cells also exhibited excessive intracellular ROS, diminished MMP, nuclear damages, and apoptosis induction in AGS cells. The enhanced expression of pro-apoptotic proteins and inhibition of PI3K/Akt/mTOR signaling pathway was noted in ZnO-Pip-NC treated cells. In vivo studies proved that the ZnO-Pip-NC noticeably restored the antioxidants in the GC animals and also prevented the gastric mucosa and inhibited the GC tumor formation. In conclusion, the findings of this investigation confirmed the anticancer potential of ZnO-Pip-NC against the GC via inhibiting the PI3K/Akt/mTOR signaling pathway.     

Iridin Induces G2/M Phase Cell Cycle Arrest and Extrinsic Apoptotic Cell Death through PI3K/AKT Signaling Pathway in AGS Gastric Cancer Cells

Iridin is a natural flavonoid found in Belamcanda chinensis documented for its broad spectrum of biological activities like antioxidant, antitumor, and antiproliferative effects. In the present study, we have investigated the antitumor potential of iridin in AGS gastric cancer cells. Iridin treatment decreases AGS cell growth and promotes G2/M phase cell cycle arrest by attenuating the expression of Cdc25C, CDK1, and Cyclin B1 proteins. Iridin-treatment also triggered apoptotic cell death in AGS cells, which was verified by cleaved Caspase-3 (Cl- Caspase-3) and poly ADP-ribose polymerase (PARP) protein expression. Further apoptotic cell death was confirmed by increased apoptotic cell death fraction shown in allophycocyanin (APC)/Annexin V and propidium iodide staining. Iridin also increased the expression of extrinsic apoptotic pathway proteins like Fas, FasL, and cleaved Caspase-8 in AGS cells. On the contrary, iridin-treated AGS cells did not show variations in proteins related to an intrinsic apoptotic pathway such as Bax and Bcl-xL. Besides, Iridin showed inhibition of PI3K/AKT signaling pathways by downregulation of (p-PI3K, p-AKT) proteins in AGS cells. In conclusion, these data suggest that iridin has anticancer potential by inhibiting PI3K/AKT pathway. It could be a basis for further drug design in gastric cancer treatment.     

Discovery of Novel Dual Extracellular Regulated Protein Kinases (ERK) and Phosphoinositide 3-Kinase (PI3K) Inhibitors as a Promising Strategy for Cancer Therapy

Concomitant inhibition of MAPK and PI3K signaling pathways has been recognized as a promising strategy for cancer therapy, which effectively overcomes the drug resistance of MAPK signaling pathway-related inhibitors. Herein, we report the scaffold-hopping generation of a series of 1H-pyrazolo[3,4-d]pyrimidine dual ERK/PI3K inhibitors. Compound 32d was the most promising candidate, with potent inhibitory activities against both ERK2 and PI3Kα which displays superior anti-proliferative profiles against HCT116 and HEC1B cancer cells. Meanwhile, compound 32d possessed acceptable pharmacokinetic profiles and showed more efficacious anti-tumor activity than GDDC-0980 and the corresponding drug combination (BVD-523 + GDDC-0980) in HCT-116 xenograft model, with a tumor growth inhibitory rate of 51% without causing observable toxic effects. All the results indicated that 32d was a highly effective anticancer compound and provided a promising basis for further optimization towards dual ERK/PI3K inhibitors.     

Targeting the PI3K/AKT/mTOR Signaling Pathway in Lung Cancer: An Update Regarding Potential Drugs and Natural Products

Lung cancer is one of the most common cancers and has a high mortality rate. Due to its high incidence, the clinical management of the disease remains a major challenge. Several reports have documented a relationship between the phosphatidylinositol-3-kinase (PI3K)/ protein kinase B (AKT)/ mammalian target of rapamycin (mTOR) pathway and lung cancer. The recognition of this pathway as a notable therapeutic target in lung cancer is mainly due to its central involvement in the initiation and progression of the disease. Interest in using natural and synthetic medications to target these signaling pathways has increased in recent years, with promising results in vitro, in vivo, and in clinical trials. In this review, we focus on the current understanding of PI3K/AKT/mTOR signaling in tumor development. In addition to the signaling pathway, we highlighted the therapeutic potential of recently developed PI3K/AKT/mTOR inhibitors based on preclinical and clinical trials.     

Neuroprotection signaling pathway of nerve growth factor and brain-derived neurotrophic factor against staurosporine induced apoptosis in hippocampal H19-7 cells

Neurotrophins protect neurons against excitotoxicity; however the signaling mechanisms for this protection remain to be fully elucidated. Here we report that activation of the phosphatidyl inositol 3 kinase (PI3K)/Akt pathway is critical for protection of hippocampal cells from staurosporine (STS) induced apoptosis, characterized by nuclear condensation and activation of the caspase cascade. Both nerve growth factor (NGF) and brain-derived growth factor (BDNF) prevent STS-induced apoptotic morphology and caspase-3 activity by upregulating phosphorylation of the tropomyosin receptor kinase (Trk) receptor. Inhibition of Trk receptor by K252a altered the neuroprotective effect of both NGF and BDNF whereas inhibition of the p75 neurotrophin receptor (p75NTR) had no effect. Impairment of the PI3K/Akt pathway or overexpression of dominant negative (DN)-Akt abolished the protective effect of both neurotrophins, while active Akt prevented cell death. Moreover, knockdown of Akt by si-RNA was able to block the survival effect of both NGF and BDNF. Thus, the survival action of NGF and BDNF against STS-induced neurotoxicity was mediated by the activation of PI3K/Akt signaling through the Trk receptor.     

Harmine Hydrochloride Mediates the Induction of G2/M Cell Cycle Arrest in Breast Cancer Cells by Regulating the MAPKs and AKT/FOXO3a Signaling Pathways

Breast cancer (BC) is one of the most common causes of death among women worldwide. Recently, interest in novel approaches for BC has increased by developing new drugs derived from natural products with reduced side effects. This study aimed to treat BC cells with harmine hydrochloride (HMH) to identify its anticancer effects and mechanisms. HMH treatment suppressed cell growth, migration, invasion, and colony formation in MCF-7 and MDA-MB-231 cells, regardless of the hormone signaling. It also reduced the phosphorylation of PI3K, AKT, and mTOR and increased FOXO3a expression. Additionally, HMH treatment increased p38 phosphorylation in MCF-7 cells and activated c-Jun N-terminal kinase (JNK) phosphorylation in MDA-MB-231 cells in a dose-dependent manner, where activated p38 and JNK increased FOXO3a expression. Activated FOXO3a increased the expression of p53, p21, and their downstream proteins, including p-cdc25, p-cdc2, and cyclin B1, to induce G2/M cell cycle arrest. Furthermore, HMH inhibited the PI3K/AKT/mTOR pathway by significantly reducing p-AKT expression in combination with LY294002, an AKT inhibitor. These results indicate that mitogen-activated protein kinases (MAPKs) and AKT/FOXO3a signaling pathways mediate the induction of cell cycle arrest following HMH treatment. Therefore, HMH could be a potential active compound for anticancer bioactivity in BC cells.     

Phosphoproteomic analysis identifies activated MET-axis PI3K/AKT and MAPK/ERK in lapatinib-resistant cancer cell line

Lapatinib, a dual inhibitor of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) tyrosine kinases, has shown promising results as a growth inhibitor of HER2-positive cancer cells in vitro. However, similar to other EGFR-targeting drugs, acquired resistance to lapatinib by HER2-positive cancer cells remains a major clinical challenge. To elucidate resistance mechanisms to EGFR/HER2-targeting agents, we performed a systematic quantitative comparison of the phosphoproteome of lapatinib-resistant (LR) human gastric cancer cells (SNU216-LR) versus parental cells (SNU216) using a titanium dioxide (TiO₂) phosphopeptide enrichment method and analysis with a Q-Exactive hybrid quadrupole-Orbitrap mass spectrometer. Biological network analysis of differentially expressed phosphoproteins revealed apparent constitutive activation of the MET-axis phosphatidylinositide 3-kinase (PI3K)/α-serine/threonine-protein kinase (AKT) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathways in SNU216-LR. Inhibition of the PI3K/AKT and MAPK/ERK signaling pathways in SNU216-LR also leads to cell cycle arrest, confirming the biological network analysis. Lapatinib sensitivity was restored when cells were treated with several molecular targeting agents in combination with lapatinib. Thus, by integrating phosphoproteomic data, protein networks and effects of signaling pathway modulation on cell proliferation, we found that SNU216-LR maintains constitutive activation of the PI3K/AKT and MAPK/ERK pathways in a MET-dependent manner. These findings suggest that pathway activation is a key compensatory intracellular phospho-signaling event that may govern gastric cancer cell resistance to drug treatment.     

Synergistic induction of cancer cell migration regulated by Gβγ and phosphatidylinositol 3-kinase

Phosphatidylinositol 3-kinase (PI3K) is essential for both G protein-coupled receptor (GPCR)- and receptor tyrosine kinase (RTK)-mediated cancer cell migration. Here, we have shown that maximum migration is achieved by full activation of phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 (P-Rex1) in the presence of Gβγ and PI3K signaling pathways. Lysophosphatidic acid (LPA)- induced migration was higher than that of epidermal growth factor (EGF)-induced migration; however, LPA-induced activation of Akt was lower than that stimulated by EGF. LPA-induced migration was partially blocked by either Gβγ or RTK inhibitor and completely blocked by both inhibitors. LPA-induced migration was synergistically increased in the presence of EGF and vice versa. In correlation with these results, sphingosine-1-phosphate (S1P)-induced migration was also synergistically induced in the presence of insulin-like growth factor-1 (IGF-1). Finally, silencing of P-Rex1 abolished the synergism in migration as well as in Rac activation. Moreover, synergistic activation of MMP-2 and cancer cell invasion was attenuated by silencing of P-Rex1. Given these results, we suggest that P-Rex1 requires both Gβγ and PI3K signaling pathways for synergistic activation of Rac, thereby inducing maximum cancer cell migration and invasion.     

Prostaglandin E2 stimulates angiogenesis by activating the nitric oxide/cGMP pathway in human umbilical vein endothelial cells

Prostaglandin E2 (PGE2), a major product of cyclooxygenase, has been implicated in modulating angiogenesis, vascular function, and inflammatory processes, but the underlying mechanism is not clearly elucidated. We here investigated the molecular mechanism by which PGE2 regulates angiogenesis. Treatment of human umbilical vein endothelial cells (HUVEC) with PGE2 increased angiogenesis. PGE2 increased phosphorylation of Akt and endothelial nitric oxide synthase (eNOS), eNOS activity, and nitric oxide (NO) production by the activation of cAMP-dependent protein kinase (PKA) and phosphatidylinositol 3-kinase (PI3K). Dibutyryl cAMP (DB-cAMP) mimicked the role of PGE2 in angiogenesis and the signaling pathway, suggesting that cAMP is a down-stream mediator of PGE2. Furthermore, PGE2 increased endothelial cell sprouting from normal murine aortic segments, but not from eNOS-deficient ones, on Matrigel. The angiogenic effects of PGE2 were inhibited by the inhibitors of PKA, PI3K, eNOS, and soluble guanylate cyclase, but not by phospholipase C inhibitor. These results clearly show that PGE2 increased angiogenesis by activating the NO/cGMP signaling pathway through PKA/PI3K/Akt-dependent increase in eNOS activity.     

Kinetic analysis of PI3K reactions with fluorescent PIP<Subscript>2</Subscript> derivatives

Phosphatidylinositol 3-kinase (PI3K) signaling plays important roles in cell differentiation, proliferation, and migration. Increased mutations and expression levels of PI3K are hallmarks for the development of certain cancers. Pharmacological targeting of PI3K activity has also been actively pursued as a novel cancer therapeutic. Consequently, measurement of PI3K activity in different cell types or patient samples holds the promise as being a novel diagnostic tool. However, the direct measurement of cellular PI3K activity has been a challenging task. We report here the characterization of two fluorescent PIP₂ derivatives as reporters for PI3K enzymatic activity. The reporters are efficiently separated from their corresponding PI3K enzymatic products through either thin layer chromatography (TLC) or capillary electrophoresis (CE), and can be detected with high sensitivity by fluorescence. The biophysical and kinetic properties of the two probes are measured, and their suitability to characterize PI3K inhibitors is explored. Both probes show similar capacity as PI3K substrates for inhibitor characterization, yet also possess distinct properties that may suggest their different applications. These characterizations have laid the groundwork to systematically measure cellular PI3K activity, and have the potential to generate molecular fingerprints for diagnostic and therapeutic applications.     

Combined Treatment with PI3K Inhibitors BYL-719 and CAL-101 Is a Promising Antiproliferative Strategy in Human Rhabdomyosarcoma Cells

Rhabdomyosarcoma (RMS) is a highly malignant and metastatic pediatric cancer arising from skeletal muscle myogenic progenitors. Recent studies have shown an important role for AKT signaling in RMS progression. Aberrant activation of the PI3K/AKT axis is one of the most frequent events occurring in human cancers and serves to disconnect the control of cell growth, survival, and metabolism from exogenous growth stimuli. In the study reported here, a panel of five compounds targeting the catalytic subunits of the four class I PI3K isoforms (p110α, BYL-719 inhibitor; p110β, TGX-221 inhibitor; p110γ, {"type":"entrez-protein","attrs":{"text":"CZC24832","term_id":"994587862","term_text":"CZC24832"}}CZC24832; p110δ, CAL-101 inhibitor) and the dual p110α/p110δ, AZD8835 inhibitor, were tested on the RMS cell lines RD, A204, and SJCRH30. Cytotoxicity, cell cycle, apoptosis, and the activation of downstream targets were analyzed. Of the individual inhibitors, BYL-719 demonstrated the most anti-tumorgenic properties. BYL-719 treatment resulted in G1/G0 phase cell cycle arrest and apoptosis. When combined with CAL-101, BYL-719 decreased cell viability and induced apoptosis in a synergistic manner, equaling or surpassing results achieved with AZD8835. In conclusion, our findings indicate that BYL-719, either alone or in combination with the p110δ inhibitor, CAL-101, could represent an efficient treatment for human rhabdomyosarcoma presenting with aberrant upregulation of the PI3K signaling pathway.     

Combination of phosphatidylinositol 3-kinases pathway inhibitor and photodynamic therapy in endothelial and tumor cells

Tumor recurrence due to incomplete eradication of tumor cells is a major problem facing current cancer therapies. To overcome this problem, it is necessary to enhance cell killing and/or prevent cell regrowth after treatment. Because phosphatidylinositol 3-kinases (PI3K) pathway plays an important role in stimulating cell survival and growth, we studied the feasibility of using a PI3K pathway inhibitor NVP-BEZ235 (BEZ235) to enhance the effectiveness of vascular-targeted photodynamic therapy (vPDT) with verteporfin. We found that BEZ235 or PDT alone significantly inhibited cell growth in both SVEC endothelial and PC-3 prostate cancer cells, although SVEC cells appeared to be more responsive than PC-3 cells. Autophagy was detected after both BEZ235 and verteporfin-PDT in both cell lines. Autophagy appeared to protect cells from PDT-induced cell death because inhibition of autophagy increased cell death. Autophagic flux assay revealed that PDT actually decreased autophagic flux especially at a high dose of verteporfin. Combination of BEZ235 and PDT caused greater inhibition of PI3K signaling pathway, leading to enhanced cell growth inhibition in both cell lines. SVEC cells exhibited a higher sensitivity towards such a combination than PC-3 cells. Our data indicated that BEZ235 in combination with PDT provides a promising approach of enhancing therapeutic response.     

Visnagin inhibits cervical cancer cells proliferation through the induction of apoptosis and modulation of PI3K/AKT/mTOR and MAPK signaling pathway

Cervical cancer, a silent killer is a second most common type of malignant tumor detected in women’s world wide. In modern medicine the usage of phytochemicals to develop drugs for treating various chronic diseases is rapidly increasing. One such phytochemical is visnagin, a furanochrome present in fruits of Ammi visnaga. We investigated the anticarcinogenic potency of visnagin against human cervical carcinoma cells. The antioxidant potency of visnagin was analyzed with FRAP assay, DPPH assay, Chemiluminscence assay and ORAC assay. The cytotoxic effect of visnagin on normal epithelial Vero cells and human cervical cancer HeLa cells were analyzed using MTT assay. The effect of visnagin on antioxidant system was examined by measuring the levels of TBARS, SOD and GSH using the colorimetric assay techniques. DCFH-DA staining, AO/EtBr staining, propidium iodide staining was performed to assess the apoptotic induction potency of visnagin against cervical cancer cells. The ability of visnagin to inhibit cancer cell migration was examined with scratch wound assay. The anticarcinogenic property of visnagin was confirmed by analyzing the gene expression of PI3K/AKT/mTOR signaling proteins and MAPK signaling proteins using qPCR analysis. Visnagin exhibited increased Trolox equivalent value in all the four antioxidant potency estimating experiments. Visnagin induced cytotoxic effect only on carcinoma cells, decreased the antioxidants and increased the generation of ROS. It also induced apoptosis and inhibited the cancer cell migration. The qPCR analysis confirms visnagin decreases the gene expression cell cycle regulating protein of both PI3K/AKT/mTOR and MAPK pathway. Overall our results authentically prove visnagin inhibits the progression of cervical cancer in vitro. Therefore it can be an ideal drug of choice which can subject to further investigation for treating cervical cancer.