Signaling through the Phosphatidylinositol 3-Kinase (PI3K)/Mammalian Target of Rapamycin (mTOR) Axis Is Responsible for Aerobic Glycolysis mediated by Glucose Transporter in Epidermal Growth Factor Receptor (EGFR)-mutated Lung Adenocarcinoma

Oncogenic epidermal growth factor receptor (EGFR) signaling plays an important role in regulating global metabolic pathways, including aerobic glycolysis, the pentose phosphate pathway (PPP), and pyrimidine biosynthesis. However, the molecular mechanism by which EGFR signaling regulates cancer cell metabolism is still unclear. To elucidate how EGFR signaling is linked to metabolic activity, we investigated the involvement of the RAS/MEK/ERK and PI3K/AKT/mammalian target of rapamycin (mTOR) pathways on metabolic alteration in lung adenocarcinoma (LAD) cell lines with activating EGFR mutations. Although MEK inhibition did not alter lactate production and the extracellular acidification rate, PI3K/mTOR inhibitors significantly suppressed glycolysis in EGFR-mutant LAD cells. Moreover, a comprehensive metabolomics analysis revealed that the levels of glucose 6-phosphate and 6-phosphogluconate as early metabolites in glycolysis and PPP were decreased after inhibition of the PI3K/AKT/mTOR pathway, suggesting a link between PI3K signaling and the proper function of glucose transporters or hexokinases in glycolysis. Indeed, PI3K/mTOR inhibition effectively suppressed membrane localization of facilitative glucose transporter 1 (GLUT1), which, instead, accumulated in the cytoplasm. Finally, aerobic glycolysis and cell proliferation were down-regulated when GLUT1 gene expression was suppressed by RNAi. Taken together, these results suggest that PI3K/AKT/mTOR signaling is indispensable for the regulation of aerobic glycolysis in EGFR-mutated LAD cells.     

The 3-deoxysappanchalcone induces ROS-mediated apoptosis and cell cycle arrest via JNK/p38 MAPKs signaling pathway in human esophageal cancer cells

BackgroundThe 3-deoxysappanchalcone (3-DSC), a chemical separated from Caesalpinia sappan L, has been substantiated to display anti-inflammatory, anti-influenza, and anti-allergy activities according to previous studies. However, the underlying mechanisms of action on esophageal cancer remain unknown.PurposeThe present research aims to survey the action mechanisms of 3-DSC in esophageal squamous cell carcinoma (ESCC) cells in vitro.MethodsEvaluation of cytotoxicity was determined by MTT tetrazolium salt assay and soft agar assay. Cell cycle distribution, apoptosis induction, reactive oxygen species (ROS) generation, mitochondrial membrane potential (MMP), and multi-caspases activity were appreciated by Muse™ Cell Analyzer. The expressions of cell cycle- and apoptosis-related proteins were presented using Western blotting.Results3-DSC blocked cell growth and colony formation ability in a concentration-dependent manner and invoked apoptosis, G2/M cell cycle arrest, ROS production, MMP depolarization, and multi-caspase activity. Furthermore, Western blotting results demonstrated that 3-DSC upregulated the expression of phospho (p)-c-jun NH2-terminal kinases (JNK), p-p38, cell cycle regulators, pro-apoptotic proteins, and endoplasmic reticulum (ER) stress-related proteins whereas downregulated the levels of anti-apoptotic proteins and cell cycle promoters. The effects of 3-DSC on ROS induction were counteracted by pretreatment with N-acetyl-L-cysteine (NAC). Also, our results indicated that p38 (SB203580) and JNK (SP600125) inhibitor slightly inhibited 3-DSC-induced apoptosis. These results showed that 3-DSC-related G2/M phase cell cycle arrest and apoptosis by JNK/p38 MAPK signaling pathway in ESCC cells were mediated by ROS.ConclusionROS generation by 3-DSC in cancer cells could be an attractive strategy for apoptosis of cancer cells by inducing cell cycle arrest, ER stress, MMP loss, multi-caspase activity, and JNK/p38 MAPK pathway. Our findings suggest that 3-DSC is a promising novel therapeutic candidate for both prevention and treatment of esophageal cancer.     

Gomisin A ameliorates metastatic melanoma by inhibiting AMPK and ERK/JNK-mediated cell survival and metastatic phenotypes

BackgroundGomisin A (G.A), a lignan compound extracted from the fruits of Schisandra chinensis, is known to exert anti-tumor effects on hepatocarcinoma and colorectal cancer cells. Suppression of proliferation and metastatic abilities of cancer cells are some effective cancer treatment methods.PurposeThe objective of this study is to investigate the effects of G.A on metastatic melanoma, and the mechanism by which it affects metastatic melanoma.Study designThe anti-proliferative and anti-metastatic effects of G.A were observed in in vitro and in vivo.MethodsWST assay and flow cytometry were conducted to investigate the effect of G.A on proliferation, cell cycle arrest, and apoptosis in metastatic melanoma cell lines. Migration and invasion abilities of G.A-treated melanoma cells were observed by wound healing and invasion assays.ResultsG.A (25–100 μM) decreased the viability of melanoma cells by inducing cell cycle arrest and apoptosis. These anti-proliferative effects of G.A were found to be mediated by AMPK, ERK, and JNK activation. G.A (5–20 μM) decreased the migration and invasion of melanoma cells by suppressing epithelial-mesenchymal transition (EMT). Consequently, G.A (2–50 mg/kg) inhibited lung metastasis by suppressing EMT and inducing cell cycle arrest and apoptosis in melanoma cells.ConclusionThese results conclude that G.A has the potential to reduce metastatic melanoma through its anti-proliferative and anti-metastatic effects.     

Ginsenoside-Rg2 affects cell growth via regulating ROS-mediated AMPK activation and cell cycle in MCF-7 cells

BackgroundGinsenoside-Rg2 (G-Rg2) is a protopanaxatriol-type ginsenoside isolated from ginseng. It has been found to exhibit various pharmacological effects, including antioxidant, anti-inflammatory, and anticancer effects.PurposeThis study aimed to investigate the anticancer effects of G-Rg2 on estrogen receptor-positive MCF-7 breast cancer (BC) cells, and the underlying mechanisms involving in reactive oxygen species (ROS) production.Study design/MethodsCell viability, cell cycle distribution, apoptosis, and ROS production were measured following exposure to G-Rg2. The protein expression levels of p-ERK1/2, p-Akt, PARP, p-Rb, cyclin D1, CDK6, and p-AMPK were quantified using western blot analysis. The in vivo activity of G-Rg2 was assessed in a xenograft model. Immunohistochemistry staining for p-Rb and p-AMPK was performed in tumor tissues.ResultsG-Rg2 significantly decreased cell viability but increased cell apoptosis. In MCF-7 cells, G-Rg2 increased ROS production by inhibiting ERK1/2 and Akt activation. G-Rg2-induced ROS induced G0/G1 cell cycle arrest and AMPK phosphorylation. In the xenograft model, the 5 mg/kg G-Rg2-treated group showed decreased tumor volume and weight, similar to the 5 mg/kg 4-OHT-treated group, compared to the control group. Immunohistochemistry staining showed that G-Rg2 treatment decreased Rb phosphorylation, while increasing AMPK phosphorylation in tumor tissues.ConclusionG-Rg2 has potential anticancer effects by increasing the ROS-AMPK signaling pathway and inhibiting ERK1/2 and Akt activation-mediated cell proliferation and cell cycle progression in MCF-7 BC cells.     

Atractylenolide I inhibits colorectal cancer cell proliferation by affecting metabolism and stemness via AKT/mTOR signaling

BackgroundAtractylenolide I (ATL-1) is a natural herbal compound used in traditional Chinese medicine that has exhibited anti-cancer properties. The anti-tumorigenic activity of ATL-1 against colorectal cancer (CRC) and the underlying signaling pathways involved in its mechanisms are examined here.HypothesisATL-1 exerts therapeutic effect against CRC by disrupting glucose metabolism and cancer stem cell maintenance via AKT/mTOR pathway regulation.Study designIn vitro studies were performed in COLO205 and HCT116 CRC cell lines and in vivo studies were conducted in a mouse xenograft model of CRC tumor.MethodsCRC cells were treated with ATL-1 at various concentrations, with or without inhibitors of AKT or mTOR. Cell proliferation, apoptosis, invasion, stemness maintenance, glucose metabolism, and AKT/mTOR signaling were evaluated. CRC tumor-xenografted mice were treated with an AKT inhibitor and/or ATL-1, and glucose metabolism and stemness maintenance were examined in tumor tissues.ResultsATL-1 significantly inhibited the invasion of CRC cells by inducing their apoptosis, possibly via the excessive production of reactive oxygen species. Glucose metabolism (Warburg effect) was also altered and stem-like traits were suppressed by ATL-1. In addition, ATL-1 effectively acted as an inhibitor or AKT/mTOR by downregulating the phosphorylation of proteins related to the AKT/mTOR pathway. In vivo studies showed that tumor weight and volume were reduced by ATL-1 and that aerobic glycolysis, stemness maintenance, and AKT/mTOR activation were impaired by ATL-1 in colorectal tumors.ConclusionsATL-1 acts as an effective agent to suppress colorectal tumor progression, mainly by inhibiting CRC cell proliferation through altering apoptosis, glucose metabolism, and stem-like behavior. These processes were mediated by the AKT/mTOR signaling pathway both in vitro and in vivo. ATL-1 may be a potential agent to be used in molecular-targeted strategies for cancer treatment.     

Longikaurin A,a natural ent-kaurane,induces G2/M phase arrest via downregulation of Skp2 and apoptosis induction through ROS/JNK/c-Jun pathway in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer, and is also highly resistant to conventional chemotherapy treatments. In this study, we report that Longikaurin A (LK-A), an ent-kaurane diterpenoid isolated from the plant Isodon ternifolius, induced cell cycle arrest and apoptosis in human HCC cell lines. LK-A also suppressed tumor growth in SMMC-7721 xenograft models, without inducing any notable major organ-related toxicity. LK-A treatment led to reduced expression of the proto-oncogene S phase kinase-associated protein 2 (Skp2) in SMMC-7721 cells. Lower Skp2 levels correlated with increased expression of p21 and p-cdc2 (Try15), and a corresponding decrease in protein levels of Cyclin B1 and cdc2. Overexpression of Skp2 significantly inhibited LK-A-induced cell cycle arrest in SMMC-7721 cells, suggesting that LK-A may target Skp2 to arrest cells at the G2/M phase. LK-A also induced reactive oxygen species (ROS) production and apoptosis in SMMC-7721 cells. LK-A induced phosphorylation of c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase and P38 MAP kinase. Treatment with, the JNK inhibitor SP600125 prevented LK-A-induced apoptosis in SMMC-7721 cells. Moreover, the antioxidant N-acetylcysteine prevented phosphorylation of both JNK and c-Jun. Taken together, these data indicate that LK-A induces cell cycle arrest and apoptosis in cancer cells by dampening Skp2 expression, and thereby activating the ROS/JNK/c-Jun signaling pathways. LK-A is therefore a potential lead compound for development of antitumor drugs targeting HCC.     

Melissa officinalis extract induces apoptosis and inhibits proliferation in colon cancer cells through formation of reactive oxygen species

PurposeEfficient strategies for the prevention of colon cancer are extensively being explored, including dietary intervention and the development of novel phytopharmaceuticals. Safe extracts of edible plants contain structurally diverse molecules that can effectively interfere with multi-factorial diseases such as colon cancer. In this study, we describe the antiproliferative and proapoptotic effects of ethanolic lemon balm (Melissa officinalis) leaves extract in human colon carcinoma cells. We further investigated the role of extra- and intracellular reactive oxygen species (ROS).MethodsAntitumor effects of lemon balm extract (LBE) were investigated in HT-29 and T84 human colon carcinoma cells. Inhibition of proliferation was analyzed by DNA quantification. The causal cell cycle arrest was determined by flow cytometry of propidium iodide-stained cells and by immunoblotting of cell cycle regulator proteins. To investigate apoptosis, cleavage of caspases 3 and 7 was detected by immunoblotting and fluorescence microscopy. Phosphatidylserine externalization was measured by Annexin V assays. Mechanistic insights were gained by measurement of ROS using the indicator dyes CM-H₂DCFDA and Cell ROX Green.ResultsAfter 3 and 4 days of treatment, LBE inhibited the proliferation of HT-29 and T84 colon carcinoma cells with an inhibitory concentration (IC₅₀) of 346 and 120 µg/ml, respectively. Antiproliferative effects were associated with a G2/M cell cycle arrest and reduced protein expression of cyclin dependent kinases (CDK) 2, 4, 6, cyclin D3, and induced expression of cyclin-dependent kinase inhibitor 2C (p18) and 1A (p21). LBE (600 µg/ml) induced cleavage of caspases 3 and 7 and phosphatidylserine externalization. LBE-induced apoptosis was further associated with formation of ROS, whereas quenching of ROS by antioxidants completely rescued the colon carcinoma cells from LBE-induced apoptosis.ConclusionsLemon balm (Melissa officinalis) extract inhibits the proliferation of colon carcinoma cells and induces apoptosis through formation of ROS. Taken together, LBE or subfractions thereof could be used for the prevention of colon cancer.     

Eldecalcitol induces apoptosis and autophagy in human osteosarcoma MG-63 cells by accumulating ROS to suppress the PI3K/Akt/mTOR signaling pathway

Eldecalcitol (ED-71) is a new type of vitamin D analog, and vitamin D has been reported to have therapeutic effects in infectious disease, autoimmune disease, and cancer. However, the anti-cancer effect of ED-71 remains unclear. The objective of this study was to explore the anti-cancer effect of ED-71 in human osteosarcoma cells and to identify the related mechanism. The CCK8 assay results showed that ED-71 inhibited MG-63 cell viability in dose and time dependent manners. Cloning and Transwell invasion assays showed that ED-71 inhibited clonal and invasion ability of MG-63 cells. Flow cytometry results showed ED-71 the G2/M cycle arrest rate, apoptosis, and intracellular ROS. Western blot was used to detect cleaved-caspase-3, Bax, Bcl-2, LC3-II/LC3-I, and P62 levels and the mTOR pathway. The increase of LC3-II and P62 indicated that ED-71 induced the formation of autophagosomes and inhibited autophagy flux. Furthermore, ED-71-induced apoptosis was weakened after adding 3-methyladenine and ED-71-induced early autophagy was weakened by caspase-3 inhibitor (Z-VAD-FMK), which indicated the two processes active each other in the presence of ED-71. Furthermore, N-acetylcysteine (NAC) pretreatment reversed the ED-71-treatment outcomes, including increased apoptosis and autophagy and inhibition of the PI3K/Akt/mTOR pathway. In conclusion, our results reveal that ED-71 induced G2/M arrest, apoptosis and autophagy in MG-63 cells by accumulating ROS to suppress the PI3K/Akt/mTOR signaling pathway     

BF12, a Novel Benzofuran,Exhibits Antitumor Activity by Inhibiting Microtubules and the PI3K/Akt/mTOR Signaling Pathway in Human Cervical Cancer Cells

BF12 [(2E)‐3‐[6‐Methoxy‐2‐(3,4,5‐trimethoxybenzoyl)‐1‐benzofuran‐5‐yl]prop‐2‐enoic acid], a novel derivative of combretastatin A‐4 (CA‐4), was previously found to inhibit tumor cell lines, with a particularly strong inhibitory effect on cervical cancer cells. In this study, we investigated the microtubule polymerization effects and apoptosis signaling mechanism of BF12. BF12 showed a potent efficiency against cervical cancer cells, SiHa and HeLa, with IC₅₀ values of 1.10 and 1.06 μm , respectively. The cellular mechanism studies revealed that BF12 induced G2/M phase arrest and apoptosis in SiHa and HeLa cells, which were associated with alterations in the expression of the cell G2/M cycle checkpoint‐related proteins (cyclin B1 and cdc2) and alterations in the levels of apoptosis‐related proteins (P53, caspase‐3, Bcl‐2, and Bax) of these cells, respectively. Western blot analysis showed that BF12 inhibited the PI3 K/Akt/mTOR signaling pathway and induced apoptosis in human cervical cancer cells. BF12 was identified as a tubulin polymerization inhibitor, evidenced by the effective inhibition of tubulin polymerization and heavily disrupted microtubule networks in living SiHa and HeLa cells. By inhibiting the PI3 K/Akt/mTOR signaling pathway and inducing apoptosis in human cervical cancer cells, BF12 shows promise for use as a microtubule inhibitor.     

Association of the ERK1/2 and p38 kinase pathways with nitric oxide-induced apoptosis and cell cycle arrest in colon cancer cells

To investigate the mechanism by which nitric oxide (NO) induces cell death in colon cancer cells, we compared two types of colon cancer cells with different p53 status: HCT116 (p53 wild-type) cells and SW620 (p53-deficient) cells. We found that S-nitrosoglutathione (GSNO), the NO donor, induced apoptosis in both types of colon cancer cells. However, SW620 cells were much more susceptible than HCT116 cells to apoptotic death by NO. We investigated the role of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 kinase on NO-induced apoptosis in both types of colon cancer cells. GSNO treatment effectively stimulated activation of the ERK1/2 and p38 kinase in both types of cells. In HCT116 cells, pretreatment with PD98059, an inhibitor of ERK1/2, or SB203580, an inhibitor of p38 kinase, had no marked effect on GSNO-induced apoptosis. However, in SW620 cells, SB203580 significantly reduced the NO-induced apoptosis, whereas PD098059 increases NO-induced apoptosis. Furthermore, we found evidence of cell cycle arrest of the G₀/G₁ phase in SW620 cells but not in HCT116 cells. Inhibition of ERK1/2 with PD098059, or of p38 kinase with SB203580, reduced the GSNO-induced cell cycle arrest of the G₀/G₁ phase in SW620 cells. We therefore conclude that NO-induced apoptosis in colon cancer cells is mediated by a p53-independent mechanism and that the pathways of ERK1/2 and p38 kinase are important in NO-induced apoptosis and in the cell cycle arrest of the G₀/G₁ phase.     

ERK1/2 inhibition enhances apoptosis induced by JAK2 silencing in human gastric cancer SGC7901 cells

Recent studies suggest JAK2 signaling may be a therapeutic target for treatment of gastric cancer (GC). However, the exact roles of JAK2 in gastric carcinogenesis are not very clear. Here, we have targeted JAK2 to be silenced by shRNA and investigated the biological functions and related mechanisms of JAK2 in GC cell SGC7901. In this study, JAK2 is commonly highly expressed in GC tissues as compared to their adjacent normal tissues (n = 75, p < 0.01). Specific down-regulation of JAK2 suppressed cell proliferation and colony-forming units, induced G2/M arrest in SGC7901 cells, but had no significant effect on cell apoptosis in vitro or tumor growth inhibition in vivo. Interestingly, JAK2 silencing-induced activation of ERK1/2, and inactivation of ERK1/2 using the specific ERK inhibitor PD98059 markedly enhanced JAK2 shRNA-induced cell proliferation inhibition, cell cycle arrest and apoptosis. Ultimately, combination of PD98059 and JAK2 shRNA significantly inhibited tumor growth in nude mice. Our results implicate JAK2 silencing-induced cell proliferation inhibition, cell cycle arrest, and ERK1/2 inhibition could enhance apoptosis induced by JAK2 silencing in SGC7901 cells.     

Daphnetin triggers ROS-induced cell death and induces cytoprotective autophagy by modulating the AMPK/Akt/mTOR pathway in ovarian cancer

BackgroundOvarian cancer is one of the most common gynecological malignancies in the world. Daphnetin (Daph) was previously reported to possess antitumor potential, but its potential and molecular mechanisms in ovarian cancer remain poorly understood.PurposeIn the current study, we aimed to explore the antitumor effect and detailed mechanisms of Daph in ovarian cancer cells.MethodsThe cytotoxic effect of Daph on ovarian cells was determined in vitro and in vivo. Cell growth, proliferation, apoptosis and ROS generation were measured by CCK8 assays, colony formation assays and flow cytometry. Western blotting was used to evaluate the related signal proteins. Immunofluorescence and transmission electron microscopy were used to evaluate markers of autophagy and autophagic flux. The antitumor effects were observed in the A2780 xenograft model. Moreover, Daph-induced autophagy was observed by enhanced LC3-II accumulation and endogenous LC3 puncta, and an autophagy inhibitor further enhanced the antitumor efficacy of Daph, which indicated that the cytoprotective role of autophagy in ovarian cancer.ResultsWe found that Daph exhibited antitumor effects by inducing ROS-dependent apoptosis in ovarian cancer, which could be reversed by N-acetyl cysteine (NAC). The AMPK/Akt/mTOR pathway was involved in Daph-mediated cytoprotective autophagy, and when Daph-mediated the expression level of AMPK and autophagy were blocked, there was robust inhibition of cell proliferation and induction of apoptosis. In addition, in the A2780 xenograft model, combined treatment with Daph and an autophagy inhibitor showed obvious synergetic effects on the inhibition of cell viability and promotion of apoptosis, without any side effects.ConclusionOur results suggest that Daph triggers ROS-induced cell apoptosis and induces cytoprotective autophagy by modulating the AMPK/Akt/mTOR pathway. Moreover, the combination of Daph and autophagy inhibitor may be a potential therapeutic strategy for ovarian cancer.     

A nanosensitizer self-assembled from oleanolic acid and chlorin e6 for synergistic chemo/sono-photodynamic cancer therapy

BackgroundSono-photodynamic therapy (SPDT) which is the combination of photodynamic therapy (PDT) and sonodynamic therapy (SDT), could exert much better anti-cancer effects than monotherapy. The combination of chemotherapy and PDT or SDT has shown great potential for cancer treatment. However, the combination of SPDT and chemotherapy for cancer treatment is rarely explored.PurposeWe utilized a natural hydrophobic anti-cancer drug oleanolic acid (OA) and a photosensitizer chlorin e6 (Ce6) through self-assembly technology to form a carrier-free nanosensitizer OC for combined chemotherapy and SPDT for cancer treatment. No studies involving using carrier-free nanomedicine for combined chemotherapy/SPDT have been reported yet.Study designAfter fully characterization of OC, the in vitro and in vivo anti-cancer activities of OC were investigated and the mechanisms of the synergistic therapeutic effects were studied.MethodsOC were synthesized through self-assembly technology and characterized by dynamic light scattering (DLS) and an atomic force microscope (AFM). Confocal microscope was used to investigate the intracellular uptake efficiency and the penetration ability of OC. The cell viability of PC9 and 4T1 cells treated with OC under laser and ultrasound (US) irradiation was determined by MTT assay. Furthermore, flow cytometry was performed to detect the reactive oxygen species (ROS) generation, loss of mitochondrial membrane potential (MMP), cell apoptosis and cell cycle arrest. Finally, the anti-tumor therapeutic efficacy of OC was investigated in orthotopic 4T1 breast tumor-bearing mouse model.ResultsOC showed an average particle size of around 100 nm with excellent light stability. OC increased more than 23 times accumulation of Ce6 in cancer cells and had strong tumor penetration ability in three-dimensional (3D) multicellular tumor spheroids (MCTSs). Compared with other therapeutic options, OC showed obvious synergistic inhibitory effects under light and US irradiation in PC9 and 4T1 cells with a significant decrease in IC₅₀ values. Mechanism studies showed that OC could generate high ROS, induce MMP loss, and cause apoptosis and cell cycle arrest. In vivo studies also approved the synergistic therapeutic effects of OC in 4T1 mouse models.ConclusionSelf-assembled carrier-free nanosensitizer OC could be a promising therapeutic agent for synergistic chemo/sono-photodynamic therapy for cancer treatment.     

Mitochondrial glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase abrogate p53 induced apoptosis in a yeast model: Possible implications for apoptosis resistance in cancer cells

BackgroundEscape from apoptosis is an important hallmark of tumor progression and drug resistance in cancer cells. It is well demonstrated that over-expression of human wtp53 in Saccharomyces cerevisiae induces apoptosis by directly targeting the mitochondria. In this study, we showed that how S.cerevisiae escaped from p53 induced apoptosis in the presence of a fermentable carbon source (sucrose), but not on non-fermentable carbon source (glycerol).MethodsMitochondrial fractions from yeast cultures grown in the presence of sucrose or glycerol with and without p53 expression were fractionated and analyzed by LC-MS/MS. Differentially expressed proteins were studied and detailed biochemical analysis for selected proteins was performed.The effect of mitochondrial HXK-2 over-expression induced by p53 in sucrose grown cells on cell survival was evaluated using gene deletion/tagging, co-localisation and mitochondrial ROS detection.ResultsWe observe that mitochondria isolated from p53 over-expressing cells accumulate Pentose phosphate Pathway (PPP) enzymes including glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) which led to enhanced mitochondrial NADPH production only when cells are cultured in sucrose but not glycerol. In contrast, mitochondria isolated from Δhxk2 p53 over-expressing cells grown in sucrose did not accumulate G6PDH and 6PGDH and resulted in defective growth.ConclusionsEnhanced association of HXK2 with the mitochondria with the concomitant accumulation of G6PDG and 6PGDH results in increased NADPH that scavenges ROS and provides resistance to apoptosis.General significanceGiven the extensive similarity of aerobic glycolysis between humans and yeast, the phenomena described here could as well be responsible for the escape of apoptosis in cancer cells.     

Impaired G2/M cell cycle arrest induces apoptosis in pyruvate carboxylase knockdown MDA-MB-231 cells

BackgroundPrevious studies showed that suppression of pyruvate carboxylase (PC) expression in highly invasive breast cancer cell line, MDA-MB-231 inhibits cell growth as a consequence of the impaired cellular biosynthesis. However, the precise cellular mechanism underlying this growth restriction is unknown.MethodsWe generated the PC knockdown (PCKD) MDA-MB-231 cells and assessed their phenotypic changes by fluorescence microscopy, proliferation, apoptotic, cell cycle assays and proteomics.ResultsPC knockdown MDA-MB-231 cells had a low percentage of cell viability in association with accumulation of abnormal cells with large or multi-nuclei. Flow cytometric analysis of annexin V-7-AAD positive cells showed that depletion of PC expression triggers apoptosis with the highest rate at day 4. The increased rate of apoptosis is consistent with increased cleavage of procaspase 3 and poly (ADP-Ribose) polymerase. Cell cycle analysis showed that the apoptotic cell death was associated with G2/M arrest, in parallel with marked reduction of cyclin B levels. Proteomic analysis of PCKD cells identified 9 proteins whose expression changes were correlated with the degree of apoptosis and G2/M cell cycle arrest in the PCKD cells. STITCH analysis indicated 3 of 9 candidate proteins, CCT3, CABIN1 and HECTD3, that form interactions with apoptotic and cell cycle signaling networks linking to PC via MgATP.ConclusionsSuppression of PC in MDA-MB-231 cells induces G2/M arrest, leading to apoptosis. Proteomic analysis supports the potential involvement of PC expression in the aberrant cell cycle and apoptosis, and identifies candidate proteins responsible for the PC-mediated cell cycle arrest and apoptosis in breast cancer cells.General significanceOur results highlight the possibility of the use of PC as an anti-cancer drug target.