Inhibition of the DNA-dependent protein kinase catalytic subunit radiosensitizes malignant glioma cells by inducing autophagy

DNA-dependent protein kinase (DNA-PK) plays a major role in the repair of DNA double-strand breaks induced by ionizing radiation (IR). Lack of DNA-PK causes defective DNA double-strand break repair and radiosensitization. In general, the cell death induced by IR is considered to be apoptotic. On the other hand, nonapoptotic cell death, autophagy, has recently attracted attention as a novel response of cancer cells to chemotherapy and IR. Autophagy is a protein degradation system characterized by a prominent formation of double-membrane vesicles in the cytoplasm. Little is known, however, regarding the relationship between DNA-PK and IR-induced autophagy. In the present study, we used human malignant glioma M059J and M059K cells to investigate the role of DNA-PK in IR-induced apoptotic and autophagic cell death. Low-dose IR induced massive autophagic cell death in M059J cells that lack the catalytic subunit of DNA-PK (DNA-PKcs). Most M059K cells, the counterpart of M059J cells in which DNA-PKcs are expressed at normal levels, survived, and proliferated although a small portion of the cells underwent apoptosis. Low-dose IR inhibited the phosphorylation of p70(S6K), a molecule downstream of the mammalian target of rapamycin associated with autophagy in M059J cells but not in M059K cells. The treatment of M059K cells with antisense oligonucleotides against DNA-PKcs caused radiation-induced autophagy and radiosensitized the cells. Furthermore, antisense oligonucleotides against DNA-PKcs radiosensitized other malignant glioma cell lines with DNA-PK activity, U373-MG and T98G, by inducing autophagy. The specific inhibition of DNA-PKcs may be promising as a new therapy to radiosensitize malignant glioma cells by inducing autophagy.     

Different G2/M accumulation in M059J and M059K cells after exposure to DNA double-strand break-inducing agents

PURPOSE: To investigate and compare the cell cycle progression in relation to cell death in the human glioma cell lines, M059J and M059K, after exposure to DNA double-strand break-inducing agents. METHODS AND MATERIALS: The M059J and M059K cells, deficient and proficient in the catalytic subunit of the DNA-dependent protein kinase, respectively, were exposed to 1 and 4 Gy of photons or accelerated nitrogen ions. In addition, M059J and M059K cells were treated with 10 and 40 mug/mL of bleomycin for 30 min, respectively. Cell cycle progression, monitored by DNA flow cytometry, was measured up to 72 h after treatment. RESULTS: M059J, but not M059K, cells displayed G(2)/M accumulation after low linear energy transfer irradiation. High linear energy transfer radiation exposure however, resulted in a substantial increase of M059K cells in the G(2)/M phase detected at 48 h. At 72 h, the number of cells in the G(2)/M phase was equivalent to its control. M059J cells accumulated mainly in S phase after high linear energy transfer irradiation. In contrast to M059K, M059J cells were still blocked at 72 h. Bleomycin induced G(2)/M accumulation for both M059J and M059K cells detected 24 h after treatment. At 48 h, the percentage of bleomycin-treated M059J cells in G(2)/M phase remained high, and the number of M059K cells had decreased to control levels. Neither cell line showed cell cycle arrest (< or =10 h) after exposure to these agents. CONCLUSION: Distinct cell cycle block and release is dependent on the complexity of the induced DNA damage and the presence of the DNA-dependent protein kinase catalytic subunit.     

Autophagic Degradation of Caspase-8 Protects U87MG Cells Against H2O2-induced Oxidative Stress

Oxidative stress induces apoptosis in many cellular systems including glioblastoma cells, with caspase-8activation was regarded as a major contribution to H2O2-induced cell death. This study focused on the roleof the autophagic protein p62 in H2O2-induced apoptosis in U87MG cells. Oxidative stress was applied withH2O2, and cell apoptosis and viability were measured with use of caspase inhibitors or autophagic mediators orsiRNA p62, GFP-p62 and GFP-p62-UBA (del) transfection. We found that H2O2 -induced U87MG cell death wascorrelated with caspase-8. To understand the role of p62 in MG132-induced cell death, the levels of p62/SQSTM1or autophagy in U87MG cells were modulated with biochemical or genetic methods. The results showed that theover-expression of wild type p62/SQSTM1 significantly reduced H2O2 induced cell death, but knockdown of p62aggravated the process. In addition, inhibition of autophagy promoted p62 and active caspase-8 increasing H2O2-induced apoptosis while induction of autophagy manifested the opposite effect. We further demonstrated thatthe function of p62/SQSTM1 required its C-terminus UBA domain to attenuate H2O2 cytotoxity by inhibitionof caspase-8 activity. Our results indicated that p62/SQSTM1 was a potential contributor to mediate caspase-8activation by autophagy in oxidative stress process.     

Role of autophagy in apoptosis induction by methylene chloride extracts of Mori cortex in NCI-H460 human lung carcinoma cells

The root of Mori cortex has traditionally been used in Korea for the treatment of cutaneous inflammation, pulmonary asthma, and congestion for thousands of years. The present study was designed to validate the anticancer effects of methylene chloride extracts of the M. cortex root (MEMC) in NCI-H460 human lung carcinoma cells. Exposure to MEMC was found to result in growth inhibition by the induction of caspase?dependent apoptosis in NCI-H460 cells, which correlated with upregulated expression of death receptor (DR)4, DR5 and FasL, downregulation of anti-apoptotic Bcl-2 and Bcl-xL expression, cleavage of Bid, and loss of mitochondrial membrane potential. In addition, autophagosomes, a characteristic finding of autophagy, and markers of autophagy, conversion of microtubule-associated protein light chain-3 (LC3)-I to LC3-II and increased beclin-1 accumulation, were observed in MEMC-treated NCI-H460 cells. Inhibition of autophagy by 3-methyladenine or LC3B small interfering (siRNA) resulted in enhanced apoptotic cell death, suggesting that MEMC-induced autophagy functions as a suppressor of apoptosis. MEMC-induced autophagy was also blocked by N-acetyl-cysteine (NAC) and catalase, indicating that H2O2 can regulate autophagy. Our data demonstrate that MEMC triggers both ROS-mediated autophagy and caspase-dependent apoptosis, and that autophagy plays a protective role against apoptotic cell death.     

An antisense oligonucleotide targeted to human Ku86 messenger RNA sensitizes M059K malignant glioma cells to ionizing radiation,bleomycin, and etoposide but not DNA cross-linking agents

Ku is a heterodimer of M(r) 70,000 and M(r) 86,000 subunits. It binds with strong affinity to DNA ends and is indispensable for nonhomologous DNA end joining (NHEJ) and V(D)J recombination. In this study, we investigated whether down-regulation of the Ku86 gene, by 2'-O-methoxyethyl/uniform phosphorothioate chimeric antisense oligonucleotides (ASOs), increases the sensitivity of the DNA-protein kinase catalytic subunit (PKcs)-proficient human glioma cell line (M059K), and its isogenic DNA-PKcs-deficient counterpart (M059J), to ionizing radiation and anticancer drugs. Transfection of these cell lines with 200 nM Ku86 antisense ASOs was associated with a specific decrease in Ku86 mRNA levels (IC(50) <25 nM; n = 3) and a concomitant rapid decrease (<10% of control) in Ku86 protein expression. Moreover, transfection of M059K cells with Ku86 antisense ASOs markedly increased cell death after treatment with ionizing radiation, bleomycin, and etoposide. However, no sensitization to the DNA cross-linking agents chlorambucil and cisplatin was observed after Ku86 antisense transfection. As expected, transfection of M059J cells with Ku86 antisense ASOs did not result in any sensitization to ionizing radiation, bleomycin, or DNA cross-linking agents, but there was a 2-fold increase in sensitivity to etoposide. Thus, our results indicate that antisense ASOs targeted against Ku86 may increase the efficacy of radiotherapy and DNA-damaging agents in tumor treatment. Furthermore, Ku86 antisense ASOs may be used to create a temporal knockout in different human cell lines to further investigate the biological roles of Ku86.     

Bcl-2 switches the type of demise from apoptosis to necrosis via cyclooxygenase-2 upregulation in HeLa cell induced by hydrogen peroxide

Bcl-2 is best known for its anti-apoptotic function in a wide variety of cell types. The objective of this study was to investigate the effects of bcl-2 on the types of cell demise in the HeLa/bcl-2 cells induced by H2O2. The HeLa cell expressed stably bcl-2 was established and defined as the HeLa/bcl-2 cell strain, while the cell transfected with the empty expression vector was defined as the HeLa/vector cell strain. MTT assay revealed that the HeLa/bcl-2 cells showed a shorter life span. BrdU incorporation assay indicated that the bcl-2 exerted anti-demise effect on the HeLa/bcl-2 cells at the low concentration of H2O2. However, at the high concentration of H2O2, the death of the HeLa/bcl-2 cells was more than that of the HeLa/vector cells. The flow cytometry demonstrated that H2O2 mainly induced apoptosis in the HeLa/vector cells and elicited necrosis in the HeLa/bcl-2 cells. The addition of celecoxib to the cells treated by H2O2 could increase apoptosis in the HeLa/vector cells and convert necrosis into apoptosis in the HeLa/bcl-2 cells. The higher levels of cellular free radical and GSH were found in the HeLa/bcl-2 cells, but not in the HeLa/vector cells. With 200 microM H2O2 challenge for 48 h, the level of the cellular free radical was increased in the both strains, while the level of the GSH was decreased in the both strains. Celecoxib could reverse the difference between the both strains led by H2O2. Western blotting showed that the expression of COX-2 was always higher in the HeLa/bcl-2 cells than in the HeLa/vector cells under the both of treated and untreated with H2O2, while the level of COX-1 was relative stable in the both strains. These results suggested that the crosstalk between the bcl-2 and the COX-2 pathways could exist, the bcl-2 might up-regulate COX-2 to modify sensitivity to the types of demise in the HeLa/bcl-2 cell.     

Human polynucleotide kinase participates in repair of DNA double-strand breaks by nonhomologous end joining but not homologous recombination

Human polynucleotide kinase (hPNK) is a bifunctional enzyme possessing a 5'-DNA kinase activity and a 3'-phosphatase activity. Studies based on cell extracts and purified proteins have indicated that hPNK can act on single-strand breaks and double-strand breaks (DSB) to restore the termini to the chemical form required for further action by DNA repair polymerases and ligases (i.e., 5'-phosphate and 3'-hydroxyl termini). These studies have revealed that hPNK can bind to XRCC4, and as a result, hPNK has been implicated as a participant in the nonhomologous end joining (NHEJ) pathway for DSB repair. We sought to confirm the role of hPNK in NHEJ in the cellular setting using a genetic approach. hPNK was stably down-regulated by RNA interference expression in M059K glioblastoma cells, which are NHEJ positive, and M059J cells, which are NHEJ deficient due to a lack of DNA-PK catalytic subunit (DNA-PKcs). Whereas depletion of hPNK significantly sensitized M059K cells to ionizing radiation, no additional sensitization was conferred to M059J cells, clearly implying that hPNK operates in the same DNA repair pathway as DNA-PKcs. On the other hand, depletion of hPNK did not increase the level of sister chromatid exchanges, indicating that hPNK is not involved in the homologous recombination DSB repair pathway. We also provide evidence that the action of hPNK in the repair of camptothecin-induced topoisomerase 1 "dead-end" complexes is independent of DNA-PKcs and that hPNK is not involved in the nucleotide excision repair pathway.     

DNA-dependent protein kinase stimulates an independently active, nonhomologous, end-joining apparatus

Double-strand breaks (DSBs) can be efficiently removed from the DNA of higher eukaryotes by nonhomologous end-joining (NHEJ). Genetic studies implicate the DNA-dependent protein kinase (DNA-PK) in NHEJ, but the exact function of this protein complex in the rejoining reaction remains to be elucidated. We compared rejoining of DNA DSBs in a human glioma cell line, M059-J, lacking the catalytic subunit of DNA-PK (DNA-PKcs), and their isogenic but DNA-PK-proficient counterpart, M059-K. In both cell lines, rejoining of DNA DSBs was biphasic, with a fast and a slow component operating with a half-life of approximately 22 min and 12 h, respectively. Deficiency in DNA-PK activity did not alter the half-times of either of these components of rejoining but increased from 17 to 72% the proportion of DNA DSB rejoining with slow kinetics. DNA DSB rejoining was nearly complete in both cell lines, and there was only a small increase in the number of unrejoined breaks in M059-J as compared with M059-K cells after 30 h of incubation. Wortmannin radiosensitized to killing M059-K cells and strongly inhibited DNA DSB rejoining. Wortmannin did not affect the radiosensitivity to killing and produced only a modest inhibition in DNA DSB rejoining in M059-J cells, suggesting that, for these end points, DNA-PK is the principal target of the drug. These observations demonstrate that DNA-PK deficiency profoundly decreases the proportion of DNA DSB rejoining with fast kinetics but has only a small effect on the fraction remaining unrejoined. We propose that in higher eukaryotes, an evolutionarily conserved, independently active, but inherently slow NHEJ pathway is stimulated 30-fold by DNA-PKcs to rapidly remove DNA DSBs from the genome. The stimulation is expected to be of local nature and the presence of DNA-PKcs in the vicinity of the DNA DSB determines whether rejoining will follow fast or slow kinetics. Structural and regulatory functions of DNA-PKcs may mediate this impressive acceleration of DNA DSB rejoining, and regions of chromatin within a certain range from this large protein may benefit from these activities. We propose the term DNA-PK surveillance domains to describe these regions.     

Bcl-2 over-expression promotes genomic instability by inhibiting apoptosis of cells exposed to hydrogen peroxide

The anti-apoptotic oncogene bcl-2 is hypothesized to increase the antioxidant status of cells, thereby protecting them from oxidative stress. In this study, we examined hydrogen peroxide (H2O2)-mediated oxidative stress in Jurkat T lymphoma cells. Over-expression of Bcl-2 did not inhibit cytotoxicity at doses of H2O2 that caused necrosis (>200 microM), but it did block cell death at apoptotic doses (<200 microM). However, these cells exhibited the same initial level of protein and lipid oxidation following exposure to H2O2 as the parental cells, indicating that the anti-apoptotic activity is not associated with general antioxidant properties. Bcl-2 expression was able to protect against secondary protein carbonyl formation, which was linked to lysosome stabilization. Assessment of micronuclei formation in cells over-expressing Bcl-2 showed evidence of increased genomic instability, consistent with the impairment of apoptosis in damaged cells. We conclude that while Bcl-2 can block cytotoxicity associated with apoptosis-inducing levels of oxidative stress, it does not protect the cells from the stress itself. Bcl-2 may promote tumourigenesis by preventing the removal of oxidatively damaged cells.     

Bag-1L is a Stress-withstand Molecule Prevents the Downregulation of Mcl-1 and c-Raf Under Control of Heat Shock Proteins in Cisplatin Treated HeLa Cervix Cancer Cells

Background: Cisplatin, a DNA damaging agent, induces apoptosis through increasing DNA fragmentation.However, identification of intrinsic resistance molecules against Cisplatin is vital to estimate the success of therapy.Bag-1 (Bcl-2-associated anthanogene) is one anti-apoptotic protein involved in drug resistance impacting ontherapeutic efficiency. Elevated levels of this protein are related with increase cell proliferation rates, motilityand also cancer development. For this reason, we aimed to understand the role of Bag-1 expression in Cisplatininducedapoptosis in HeLa cervix cancer cells. Cisplatin decreased cell viability in time- and dose-dependentmanner in wt and Bag-1L+HeLa cells. Although, 10μM Cisplatin treatment induced cell death within 24h byactivating caspases in wt cells, Bag-1L stable transfection protected cells against Cisplatin treatment. To assess thepotential protective role of Bag-1, we first checked the expression profile of interacting anti-apoptotic partners ofBag-1. We found that forced Bag-1L expression prevented Cisplatin-induced apoptosis through acting on Mcl-1expression, which was reduced after Cisplatin treatment in wt HeLa cells. This mechanism was also supportedby the regulation of heat shock protein (Hsp) family members, Hsp90 and Hsp40, which were involved in theregulation Bag-1 interactome including several anti-apoptotic Bcl-2 family members and c-Raf.     

DNA double-strand break rejoining in human follicular lymphoma and glioblastoma tumor cells

Follicle center cell lymphoma is among the most radioresponsive of human cancers. To assess whether this radioresponsiveness might be a result of a compromised ability of the tumor cells to accomplish the biologically-effective repair of DNA double-strand breaks (DSBs), we have measured i) the extent of the mechanical rejoining of radiation-induced DSBs in biopsy-derived follicle center cell lymphoma cells and ii) the fidelity with which nuclear protein extracts from these cells rejoin restriction enzyme-induced DSBs. Cell suspensions derived from two lymphoma biopsies, designated FCL1 and FCL2, as well as two established human glioblastoma cell lines, M059J and M059K, were exposed to 30 Gy of gamma-rays and evaluated for their ability to rejoin DSBs using a Southern transfer-pulsed-field gel electrophoresis assay. The fidelity of rejoining of restriction enzyme-induced DSBs was assessed using a cell-free plasmid reactivation assay. Both lymphoma suspensions rejoined DSBs relatively slowly and exhibited a similar phenotype to the known DSB-rejoining deficient M059J line. The level of DSB mis-rejoining in the cell-free plasmid reactivation assay was also similar in M059J and FCL2 cells and was considerably ( approximately 6-fold) higher than in M059K cells. Because of insufficient numbers of cells, we were unable to perform this assay with the FCL1 lymphoma. These limited data suggest that follicle center cell lymphoma cells may be intrinsically deficient in performing the biologically-effective rejoining of DSBs. Such a deficiency might contribute to the radioresponsiveness of this disease and may be exploitable in the development of improved treatment strategies, such as radioimmunotherapy.     

MiR-15a and miR-16 induce autophagy and enhance chemosensitivity of Camptothecin

It has been reported that persistent or excessive autophagy promotes cancer cell death during chemotherapy, either by enhancing the induction of apoptosis or mediating autophagic cell death. Here, we show that miR-15a and miR-16 are potent inducers of autophagy. Rictor, a component of mTORC2 complex, is directly targeted by miR-15a/16. Overexpression of miR-15a/16 or depletion of endogenous Rictor attenuates the phosphorylation of mTORC1 and p70S6K, inhibits cell proliferation and G1/S cell cycle transition in human cervical carcinoma HeLa cells. Moreover, miR-15a/16 dramatically enhances anticancer drug camptothecin (CPT)-induced autophagy and apoptotic cell death in HeLa cells. Collectively, these data demonstrate that miR-15a/16 induced autophagy contribute partly to their inhibition of cell proliferation and enhanced chemotherapeutic efficacy of CPT.     

Rose bengal acetate photodynamic therapy-induced autophagy

Photodynamic therapy (PDT), an anticancer therapy requiring the exposure of cells or tissue to a photosensitizing drug followed by irradiation with visible light of the appropriate wavelength, induces cell death by the efficient induction of apoptotic as well as non-apoptotic mechanisms, such as necrosis and autophagy, or a combination of all three mechanisms. However, the exact role of autophagy in photodynamic therapy is still a matter of debate. To understand the role of autophagy in PDT, we investigated the induction of autophagy in HeLa cells photosensitized with Rose Bengal Acetate (RBAc). After incubation with Rose Bengal Acetate (10-5 M), HeLa cells were irradiated for 90 seconds (green LED DPL 305, emitting at 530 +15 nm to obtain 1.6 J/cm2 as the total light dose) and allowed to recover for 72 h. Induction of autophagy and apoptosis were observed with peaks at 8 h and 12 h after irradiation, respectively. Autophagy was detected by biochemical (Western Blotting for the LC3B protein) and morphological criteria (TEM, cytochemistry). In addition, the pan-caspase inhibitor, z-VAD, was unable to completely prevent cell death. The simultaneous onset of apoptosis and autophagy following Rose Bengal Acetate PDT is of remarkable interest in light of the findings that autophagy can result in the class II presentation of antigens and thus, explain why low dose PDT can yield anti-tumor immune responses.     

The PPARgamma ligands PGJ2 and rosiglitazone show a differential ability to inhibit proliferation and to induce apoptosis and differentiation of human glioblastoma cell lines

Peroxisome proliferator-activated receptor gamma (PPARgamma) is involved in the control of cell proliferation, apoptosis and differentiation in various tumor cells. Among PPARgamma ligands, 15-deoxy-Delta12,14-prostaglandin J2 (PGJ2), the ultimate metabolite of PGD2, plays a role in the biology of brain tumors. It is still unclear to which extent the anti-proliferative and differentiation-promoting activity of PGJ2 is mediated through PPARgamma. We compared the effects of PGJ2 with those of rosiglitazone - the synthetic agonist with the highest affinity for PPARgamma - in 4 human glioblastoma cell lines (A172, U87-MG, M059K, M059J). All cell lines expressed high levels of PPARgamma, consistent with the high levels of PPARgamma protein in 5 tumor samples. Both PGJ2 and rosiglitazone inhibited proliferation of all cell lines with a G2/M arrest and apoptosis, but only PGJ2 up-regulated p21Cip/WAF1. The growth inhibitory effect was partially reversed by the PPARgamma antagonist GW9662. We studied the time sequence of selected molecular events, that lead glioblastoma cells to apoptosis and/or differentiation, after treatment with both agonists. M059K cells committed to undergo apoptosis by PGJ2, initially up-regulated PPARgamma, and then down-regulated PPARgamma as they began apoptosis. Apoptotic cells also increased their expression of retinoic acid receptor beta (RARbeta) and retinoid X receptor alpha (RXRalpha). PGJ2 increased expression of glial fibrillary acidic protein (GFAP) and decreased levels of vimentin, structural proteins modulated during astrocytic differentiation. Unexpectedly, PGJ2 up-regulated the expression of cyclooxygenase-2 (COX-2). Rosiglitazone caused the same pattern of PPARgamma, RARbeta and RXRalpha expression as PGJ2, but no significant modulation of p21Cip/WAF1, cytoskeletal proteins or COX-2 occurred. Our data indicate that PGJ2, and rosiglitazone suppress cell proliferation and cause apoptosis in glioblastoma cell lines, most likely through a PPARgamma-dependent pathway. By contrast, the modulation of differentiation-associated proteins by PGJ2, but not rosiglitazone, suggests that PGJ2 promotes differentiation of glioblastoma cells independently of PPARgamma activation.     

The relationship between the down-regulation of DNA-PKcs or Ku70 and the chemosensitization in human cervical carcinoma cell line HeLa

The aim of this study was to clarify the function of non-homologous end-joining (NHEJ) in tumorigenesis and chemoresistance, and to explore the potential of DNA-PK as a target of reversal of chemoresistance and enhancing the sensitivity of cells to chemotherapeutic agents. Plasmid vectors pSIREN-Ku70shRNA and pSIREN-DNA-PKcssh-RNA, which coded small interfering RNA of Ku70 and DNA-PKcs, were constructed and transfected into human cervical cancer cell line HeLa. The relationship between the down-regulation of Ku70 or DNA-PKcs and tumor cell proliferation and the sensitivity of cells to chemotherapeutic agents were analyzed. Down-regulation of Ku70 and DNA-PKcs expression inhibited cell proliferation, and increased cell apoptosis in DDP-treated HeLa cells. DNA-PK might play an important role in drug resistance, and inhibition of the DNA-PK expression suppressed the growth of tumor cells and enhanced the sensitivity of cells to chemotherapeutic agents.     

Expression of calbindin-D28k is inversely correlated with proapototic gene expression in hydrogen peroxide-induced cell death in endometrial cancer cells

Calbindin-D28k (CaBP-28k) is a calcium binding protein important for intracellular Ca2+ buffering and known to have anti-apoptotic properties in neurons, osteoblasts and male germ cells. Although endometrial cancer is a common invasive gynecologic malignancy, the involvement of uterine CaBP-28k in apoptotic signaling of endometrial cancer is poorly understood. The present study investigates the role of CaBP-28k in hydrogen peroxide (H2O2)-induced apoptotic signaling in human endometrial Ishikawa cells. The dose- and time-dependent effect of H2O2 on Bax, p53 and Bcl-2 expression was assessed by Western blot analysis. Treatment of cells with 1 mM H2O2 for 1 h induced an increase in Bax and p53 expression, but the expression of Bcl-2 was not affected by H2O2 treatment. Interestingly, overexpression of CaBP-28k inhibited cell death and caused a decrease in Bax, p53 and caspase 3 expression during H2O2-induced apoptosis, suggesting that CaBP-28k blocks the up-regulation of apoptosis-related gene expression. siRNA knockdown of CaBP-28k resulted in an elevation of H2O2-induced cell death and an increase in Bax, p53 and caspase 3, providing additional evidence that induction of the CaBP-28k gene might be associated with survival signaling during H2O2-mediated cell death. Overall, these results suggest that CaBP-28k expression is inversely correlated with pro-apoptotic gene expression in human endometrial Ishikawa cells.     

Furanonaphthoquinones cause apoptosis of cancer cells by inducing the production of reactive oxygen species by the mitochondrial voltage-dependent anion channel

The mitochondrial production of reactive oxygen species has been implicated in the anticancer activity of furanonaphthoquinone. However, the mechanism of the activation remains elusive. In the current study, we found that treatment of HeLa cells with 2-methyl-5(or -8)-hydroxy-furanonaphthoquinone (FNQ13) induces mitochondrial swelling, followed by apoptosis. This toxic effect of FNQ13 was reduced by the radical scavengers alpha-tocopherol and trolox. Cytochemical experiments in isolated mitochondria showed that a combination of FNQ13 and NADH induces the production of H(2)O(2) at the exterior mitochondrial membrane surface. This production of H(2)O(2) was reduced by an antibody to the voltage-dependent anion channel (VDAC). Overexpression of the VDAC by transfection with vdac1 cDNA increased the production of H(2)O(2) by HeLa cells, whereas transfection with a small interfering RNA to VDAC reduced FNQ13-induced H(2)O(2) production and cell death due to an almost complete knockdown of VDAC expression. We also found significant correlations between the expression of VDAC and the induction of H(2)O(2) production and cell death by FNQ13 in 11 human cancer cell lines. These results indicate that the anticancer activity of furanonaphthoquinones depends on the production of reactive oxygen species by mitochondrial permeability transition pores including the VDAC.     

自噬对宫颈癌细胞增殖和迁移能力的影响

目的 观察自噬对宫颈癌细胞增殖迁移能力的影响,并探讨其可能机制。方法 采用不同浓度雷帕霉素诱导人宫颈癌HeLa细胞,吖啶橙染色观察自噬小体的形成,Western blot检测自噬相关基因LC3B以及PI3K/Akt/mTOR通路蛋白的表达情况,采用RTCA仪器实时检测细胞的增殖能力,Transwell小室检测细胞的迁移能力。应用自噬抑制剂3-methyladenine(3-MA)阻断宫颈癌细胞自噬后,采用RTCA仪器实时检测细胞的增殖能力,Transwell小室检测细胞的迁移能力。构建带有绿色荧光蛋白的LC3B质粒,转入HeLa细胞,通过G418药物筛选转染阳性细胞,荧光显微镜观察LC3B的细胞定位,Western blot检测LC3B表达。RTCA仪器实时检测野生型和LC3B过表达HeLa细胞的增殖能力,Western blot检测PI3K/Akt/mTOR通路蛋白的表达。结果 雷帕霉素诱导后,宫颈癌HeLa细胞自噬水平增高,增殖和迁移能力有所下降;PI3K/Akt/mTOR通路蛋白表达改变(P＜0.05)。自噬抑制剂3-MA诱导HeLa细胞增殖和迁移水平下降(P＜0.05)。荧光显微镜和Western blot结果显示构建的LC3质粒成功转入Hela细胞中并且能够抑制宫颈癌细胞的增殖(P＜0.05)。结论 在一定范围内,随着自噬水平的升高,HeLa细胞增殖和迁移能力降低,可能是通过PI3K/Akt/mTOR信号通路发挥作用。通过基因靶向扰乱自噬关键基因(如LC3等)而改变宫颈癌细胞自噬水平可能为宫颈癌的治疗带来新策略。     

Hydrogen peroxide controls Akt activity via ubiquitination/degradation pathways

Akt is a well-established protein that regulates cell growth, survival and anti-apoptotic mechanisms. In this study, we demonstrated that hydrogen peroxide (H2O2)-induced oxidative stress regulates the activity of the anti-apoptotic protein Akt via the ubiquitin-proteasome degradation system. H2O2 induced cytotoxicity in PC12 cells and decreased the cellular protein and phosphorylation levels of Akt in a concentration- and exposure time-dependent manner. This downregulation was blocked by the proteasome inhibitor MG132 and the Akt-specific inhibitor LY294002. In addition, an in?vivo ubiquitination assay revealed that the degradation of Akt was mediated by the ubiquitin-mediated proteasome pathway and further demonstrated that this ubiquitination was dependent on the phosphorylation status of Akt. Furthermore, the exogenously overexpressed active form of Akt, but not its inactive form, induced resistance to H2O2-mediated cell death. These results suggested that H2O2-induced cytotoxicity was mediated by active Akt degradation.