Let-7g Upregulation Attenuated the KRAS-PI3K-Rac1-Akt Axis-Mediated Bioenergetic Functions.

The aberrant activation of signaling pathways contributes to cancer cells with metabolic reprogramming. Thus, targeting signaling modulators is considered a potential therapeutic strategy for cancer. Subcellular fractionation, coimmunoprecipitation, biochemical analysis, and gene manipulation experiments revealed that decreasing the interaction of kirsten rat sarcoma viral oncogene homolog (KRAS) with p110α in lipid rafts with the use of naringenin (NGN), a citrus flavonoid, causes lipid raft-associated phosphatidylinositol 3-kinase (PI3K)-GTP-ras-related C3 botulinum toxin substrate 1 (Rac1)-protein kinase B (Akt)-regulated metabolic dysfunction of glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), leading to apoptosis in human nasopharyngeal carcinoma (NPC) cells. The use of lethal-7g (let-7g) mimic and let-7g inhibitor confirmed that elevated let-7g resulted in a decrease in KRAS expression, which attenuated the PI3K-Rac1-Akt-BCL-2/BCL-xL-modulated mitochondrial energy metabolic functions. Increased let-7g depends on the suppression of the RNA-specificity of monocyte chemoattractant protein-induced protein-1 (MCPIP1) ribonuclease since NGN specifically blocks the degradation of pre-let-7g by NPC cell-derived immunoprecipitated MCPIP1. Converging lines of evidence indicate that the inhibition of MCPIP1 by NGN leads to let-7g upregulation, suppressing oncogenic KRAS-modulated PI3K-Rac1-Akt signaling and thereby impeding the metabolic activities of aerobic glycolysis and mitochondrial OXPHOS.

Oxidative Stress-Induced Unscheduled CDK1-Cyclin B1 Activity Impairs ER-Mitochondria-Mediated Bioenergetic Metabolism.

Targeting the activities of endoplasmic reticulum (ER)-mitochondrial-dependent metabolic reprogramming is considered one of the most promising strategies for cancer treatment. Here, we present biochemical subcellular fractionation, coimmunoprecipitation, gene manipulation, and pharmacologic evidence that induction of mitochondria-localized phospho (p)-cyclin dependent kinase 1 (CDK1) (Thr 161)-cyclin B1 complexes by apigenin in nasopharyngeal carcinoma (NPC) cells impairs the ER-mitochondrial bioenergetics and redox regulation of calcium (Ca++) homeostasis through suppressing the B cell lymphoma 2 (BCL-2)/BCL-2/B-cell lymphoma-extra large (BCL-xL)-modulated anti-apoptotic and metabolic functions. Using a specific inducer, inhibitor, or short hairpin RNA for acid sphingomyelinase (ASM) demonstrated that enhanced lipid raft-associated ASM activity confers alteration of the lipid composition of lipid raft membranes, which leads to perturbation of protein trafficking, and induces formation of p110α free p85α-unphosphorylated phosphatase and tensin homolog deleted from chromosome 10 complexes in the lipid raft membranes, causing disruption of phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt)-GTP-ras-related C3 botulinum toxin substrate 1 (Rac1)-mediated signaling, thus triggering the p-CDK1 (Thr 161))-cyclin B1-mediated BCL-2 (Thr 69/Ser 87)/BCL-xL (Ser 62) phosphorylation and accompanying impairment of ER-mitochondria-regulated bioenergetic, redox, and Ca++ homeostasis. Inhibition of apigenin-induced reactive oxygen species (ROS) generation by a ROS scavenger N-acetyl-L-cysteine blocked the lipid raft membrane localization and activation of ASM and formation of ceramide-enriched lipid raft membranes, returned PI3K-Akt-GTP-Rac1-modulated CDK1-cyclin B1 activity, and subsequently restored the BCL-2/BCL-xL-regulated ER-mitochondrial bioenergetic activity. Thus, this study reveals a novel molecular mechanism of the pro-apoptotic activity of ASM controlled by oxidative stress to modulate the ER-mitochondrial bioenergetic metabolism, as well as suggests the disruption of CDK1-cyclin B1-mediated BCL-2/BCL-xL oncogenic activity by triggering oxidative stress-ASM-induced PI3K-Akt-GTP-Rac1 inactivation as a therapeutic approach for NPC.

Crude extract of Rheum palmatum L. Induces cell cycle arrest S phase and apoptosis through mitochondrial-dependent pathways in U-2 OS human osteosarcoma cells.

Cancer is the second cause of death in children. Osteosarcoma is the most common primary malignancy of solid bone cancer primarily affecting adolescents and young adults. In the Chinese population, the crude extract of Rheum palmatum L. (CERP) has been used for treating different diseases, including SARS, rheumatoid arthritis, coxsackievirus B3, and human colon cancer cell, pancreatic cancer. There are no reports on CERP and human osteosarcoma cells. The present study examined effects of CERP on cytotoxicity including cell cycle distribution and cell death (apoptosis) in U-2 OS human osteosarcoma cells. CERP significantly induced S phase arrest in U-2 OS cells in a dose-dependent. CERP produced DNA damage and DNA condensation. Other effects of CERP were stimulation of ROS and Ca(2+) , mitochondria impairment, and activation of caspase-3, -8, and -9. CERP increased the levels of Bax, Bak, Bad, cyclin B, Fas, PARP, GRP78, GADD153, AIF, Endo G, Calpain-2, p21, and p27, but decreased the levels of Bcl-2, BCL-X, XIAP, Akt, CDC25A, CDK2, Cyclin A, and Cyclin E of U-2 OS cells. It was also observed that CERP promoted the expression of AIF, Endo G, GADD153, and cytochrome c. These results indicate that CERP has anticancer effects in vitro and provide the foundation for in vivo studies of animal models of osteosarcoma. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 957-969, 2016.

Chloroform Extract of Solanum lyratum Induced G0/G1 Arrest via p21/p16 and Induced Apoptosis via Reactive Oxygen Species, Caspases and Mitochondrial Pathways in Human Oral Cancer Cell Lines.

Solanum lyratum (SLEC) Thunberg (Solanaceae) has been used as a traditional herbal medicine in China for centuries. Numerous studies have shown that SLEC Thunberg (Solanaceae) extract inhibited cancer cell growth in vitro. Herein, we investigated cell death-induced by EcoAc, water, chloroform, butanol extract of SLEC in human oral cancer cell lines (HSC-3, SAS, and CAL-27) in vitro. Different SLEC extract induced cytotoxic effects in human oral cancer cells were examined by contrast phase microscopy. We selected the chloroform extract of SLEC to examine the cytotoxic effects by using DAPI staining, comet assays, flow cytometric assay, Western blotting and examination of confocal laser microscopy. SLEC decreased the percentage of viable cells, induced G0/G1 arrest and apoptosis. These effects were concentration- and time-dependent manners. SLEC increased protein levels of p21, p16, CDK2, and cyclin D1 in HSC-3, SAS, and CAL-27 cells. Also, SLEC increased CDK6 in HSC-3 and CAL-27 cells, but inhibited CDK6 in SAS cells. Cyclin E in HSC-3 and SAS cells was increased by SLEC, but it was inhibited in CAL-27 cells. SLEC suppressed the anti-apoptotic proteins Bcl-2 and Bcl-xl, but increased the pro-apoptotic proteins Bax and Bad in HSC-3, SAS, and CAL-27 cells. SLEC promoted the production of reactive oxygen species (ROS) and Ca²âº, decreased the mitochondrial membrane potential (Δψm) and stimulated NO production in HSC-3, SAS, and CAL-27 cells. Specific caspase inhibitors (caspase-8 inhibitor: Z-IETD-FMK; caspase-9 inhibitor: Z-LEHD-FMK and caspase-3 inhibitor: Z-DEVD-FMK) for caspase-8, -9, and -3 blocked SLE-activated caspase-8, -9, and -3 activities which were associated with an increase in the percentage of viable cells. Taken together, SLE induced G0/G1 arrest and apoptosis via extrinsic- and intrinsic-dependent pathways in HSC-3, SAS, and CAL-27 cells.

Cantharidin induces DNA damage and inhibits DNA repair-associated protein expressions in TSGH8301 human bladder cancer cell.

Cantharidin is an active component of mylabris, which has been used as a traditional Chinese medicine. Cantharidin has been shown to have antitumor activity against several types of human cancers in vitro and in animal models in vivo. We investigated whether cantharidin induces DNA damage and affects DNA damage repair-associated protein levels in TSGH8301 human bladder cancer cells. Using flow cytometry to measure viable cells, cantharidin was found to reduce the number of viable cells in a dose-dependent manner. Comet assay, 4',6-diamidino-2-phenylindole (DAPI) staining and DNA gel electrophoresis were used to measure DNA damage and condensation; the results indicated that cantharidin induced DNA damage (comet tail), DNA condensation (white DAPI staining) and DNA damage (DNA smear). Results from western blotting showed that cantharidin inhibited the expression of DNA-dependent serine/threonine protein kinase, poly-ADP ribose polymerase, phosphate-ataxia-telangiectasia and RAD3-related, O-6-methylguanine-DNA methyltransferase, breast cancer susceptibility protein 1, mediator of DNA damage checkpoint protein 1, phospho-histone H2A.X, but increased that of phosphorylated p53 following 6 and 24 h treatment. Confocal laser microscopy was used to examine the protein translocation; cantharidin suppressed the levels of p-H2A.X and MDC1 but increased the levels of p-p53 in TSGH8301 cells. In conclusion, we found that cantharidin-induced cell death may occur through the induction of DNA damage and suppression of DNA repair-associated protein expression in TSGH8301 cells.

An experimental study on the antileukemia effects of gypenosides in vitro and in vivo.

PURPOSE: Gypenosides (Gyp), found in Gynostemma pentaphyllum Makino, have been used as folk medicine for centuries and have exhibited diverse pharmacological effects, including antileukemia effects in vitro and in vivo. In the present study, Gyp were used to examine effects on cell viability, cell cycle, and induction of apoptosis in vitro. They were administered in the diet to mice injected with WEHI-3 cells in vivo. EXPERIMENTAL DESIGN: Effects of Gyp on WEHI-3 cells were determined by flow cytometric assay and Western blotting. RESULTS: Gyp inhibited the growth of WEHI-3 cells. These effects were associated with the induction of G0/G1 arrest, morphological changes, DNA fragmentation, and increased sub-G1 phase. Gyp promoted the production of reactive oxygen species, increased Ca(2+) levels, and induced the depolarization of the mitochondrial membrane potential. The effects of Gyp were dose and time dependent. Moreover, Gyp increased levels of the proapoptotic protein Bax, reduced levels of the antiapoptotic proteins Bcl-2, and stimulated release of cytochrome c, AIF (apoptosis-inducing factor), and Endo G (endonuclease G) from mitochondria. The levels of GADD153, GRP78, ATF6-α, and ATF4-α were increased by Gyp, resulting in ER (endoplasmic reticular) stress in WEHI-3 cells. Oral consumption of Gyp increased the survival rate of mice injected with WEHI-3 cells used as a mouse model of leukemia. CONCLUSIONS: Results of these experiments provide new information on understanding mechanisms of Gyp-induced effects on cell cycle arrest and apoptosis in vitro and in an in vivo animal model.

Berberine induced down-regulation of matrix metalloproteinase-1, -2 and -9 in human gastric cancer cells (SNU-5) in vitro.

Berberine, a yellow benzylisoquinoline alkaloid, is a constituent of Coptis chines and is commonly used in Chinese herbal medicine for patients with gastrointestinal disorders. The pharmacological effects of berberine include anti-inflammation, antidiarrhetic, antimalarial, and even antimicrobial activities. However, its mechanism of action on the cell migration of human gastric cancer SNU-5 cells is not fully understood. The effects of berberine on the percentage of viable cells were examined first and it was found that berberine induced dose-dependent inhibition in human gastric cancer SNU-5 cells. The effect of berberine on the levels of reactive oxygen species (ROS) and matrix metalloproteinase-1, -2, -7 and -9 was then examined using Western blotting and the results showed that berberine induced ROS production for up to 6 hours of incubation. It was also found that berberine induced downregulation of MMP-1 -2, and -9 but did not affect the level of MMP-7. The mRNA levels of MMPs in SNU-5 cells after treatment with berberine for 24 hours were investigated using a polymerase chain reaction and the results showed that berberine inhibited the gene expression of MMP-1, -2 and -9 in human SNU-5 cells but it did not affect MMP-7. In conclusion, berberine appears to exert its anticancer properties by inducing ROS production and prevention of cell migration via inhibition of the gene expression of MMP-1, -2 and -9 in human gastric cancer SNU-5 cancer cells.