Xiujuan Qu

Gastric cancer exosomes promote tumour cell proliferation through PI3K/Akt and MAPK/ERK activation

BACKGROUND Exosomes are nanometer-sized vesicles that are released by normal and neoplastic cells. Previous studies have focused on the interaction between tumour-derived exosomes and the immune system, as a consequence of immune suppression or enhancement. However, the effects of tumour-derived exosomes on tumour cells themselves have not been well studied. AIMS To investigate the effects of gastric cancer exosomes on tumour cell proliferation and the possible mechanisms. METHODS By serial centrifugation and sucrose gradient ultracentrifugation, we isolated and purified the exosomes from gastric cancer SGC7901 cells, then viewed them by electron microscopy. Cell proliferation was measured by 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide assay. Protein expression was assayed by Western blotting. RESULTS SGC7901-cell-derived exosomes promoted the proliferation of SGC7901 and BGC823 cells. The increase in proliferation induced by exosomes was accompanied by activation of Akt and extracellular-regulated protein kinase, and phosphoinositide 3-kinase or extracellular-regulated protein kinase inhibitor partially reversed the proliferative effect of exosomes. Moreover, the exosome-induced increase in activity of Akt and extracellular-regulated protein kinase coincided with decreased expression of the Casitas B-lineage lymphoma family of ubiquitin ligases. CONCLUSION Gastric cancer exosomes promoted tumour cell proliferation, at least in part, by activation of PI3K/Akt and mitogen-activated protein kinase/extracellular-regulated protein kinase pathways. The decreased expression of Casitas B-lineage lymphoma proteins might have contributed to the activation of Akt and extracellular-regulated protein kinase.

PI3K/Akt is involved in bufalin-induced apoptosis in gastric cancer cells.

Bufalin is the active ingredient of the Chinese medicine Chan Su, and it has been reported that bufalin induces apoptosis in some human leukemia and solid cancer cell lines. The exact mechanism of bufalin-induced apoptosis is, however, still not clear. In this study, we demonstrated that bufalin inhibited the proliferation of gastric cancer MGC803 cells in a dose-dependent and time-dependent manner. At a low concentration (20 nmol/l), bufalin induced M-phase cell cycle arrest, whereas at a high concentration (80 nmol/l) it induced apoptosis in MGC803 cells. Bufalin increased the Bax/Bcl-2 ratio and activated caspase-3 during the apoptotic process of MGC803 cells. It should be noted that bufalin transiently activated the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway and then inhibited it completely, and upregulated the Casitas B-lineage lymphoma (Cbl) family of ubiquitin ligases, upstream modulators of PI3K. A combination of bufalin and LY294002, a PI3K-specific inhibitor, enhanced apoptosis, but PD98059, an extracellular-regulated protein kinase-specific inhibitor, had no significant effect on bufalin-induced apoptosis. These results suggested that the PI3K/Akt pathway might play a key role in bufalin-induced apoptosis in gastric cancer MGC803 cells.

β-Elemene-induced autophagy protects human gastric cancer cells from undergoing apoptosis

Backgroundβ-Elemene, a compound found in an herb used in traditional Chinese medicine, has shown promising anti-cancer effects against a broad spectrum of tumors. The mechanism by which β-elemene kills cells remains unclear. The aim of the present study is to investigate the anti-tumor effect of β-elemene on human gastric cancer cells and the molecular mechanism involved.Resultsβ-Elemene inhibited the viability of human gastric cancer MGC803 and SGC7901 cells in a dose-dependent manner. The suppression of cell viability was due to the induction of apoptosis. A robust autophagy was observed in the cells treated with β-elemene; it was characterized by the increase of punctate LC3 dots, the cellular morphology, and the increased levels of LC3-II protein. Further study showed that β-elemene treatment up-regulated Atg5-Atg12 conjugated protein but had little effect on other autophagy-related proteins. PI3K/Akt/mTOR/p70S6K1 activity was inhibited by β-elemene. Knockdown of Beclin 1 with small interfering RNA, or co-treatment with the autophagy inhibitor, 3-methyladenine or chlorochine enhanced significantly the antitumor effects of β-elemene.ConclusionsOur data provides the first evidence that β-elemene induces protective autophagy and prevents human gastric cancer cells from undergoing apoptosis. A combination of β-elemene with autophagy inhibitor might thus be a useful therapeutic option for advanced gastric cancer.

Arsenic trioxide induces apoptosis and G2/M phase arrest by inducing Cbl to inhibit PI3K/Akt signaling and thereby regulate p53 activation

Arsenic trioxide (ATO) strongly induces apoptosis in acute promyelocytic leukemia (APL), but it induces cell cycle arrest in most solid tumors. In this study, we investigated the mechanism of ATO action on APL-derived NB4 cells and gastric cancer cell lines. ATO decreased the viability of both cell lines, but gastric cancer cells were much less susceptible. ATO-induced G2/M phase arrest and p53 degradation in gastric cancer MGC803 cells. In contrast, ATO-induced apoptosis in NB4 cells without degradation of p53. Both processes were accompanied by transient activation of Akt. The PI3K/Akt inhibitor LY294002 significantly increased the amount of p53 protein and ATO-induced apoptosis in both cell lines and decreased G2/M phase arrest of MGC803 cells. In addition, ATO up-regulated the expression of Cbl proteins in both cell lines. Inhibition of Cbl with the proteasome inhibitor Ps341 decreased apoptosis in NB4 cells and increased the G2/M phase arrest of MGC803 cells, and it also prolonged the activation of PI3K/Akt by ATO. Consistent results with those in MGC803 cells were showed in gastric cancer cell BGC823 and SGC7901 after ATO treatment. These results demonstrate that inhibition of PI3K/Akt signaling by Cbl is involved in both ATO-induced apoptosis of NB4 cells and ATO-induced G2/M phase arrest of gastric cancer cells. Cbl achieved these effects probably via its regulating PI3K/Akt pathway, and thereby modulated p53 activation.

Oxaliplatin enhances TRAIL-induced apoptosis in gastric cancer cells by CBL-regulated death receptor redistribution in lipid rafts

Tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) is a member of the tumor necrosis factor family that selectively induces apoptosis in cancer cells. However, gastric cancer cells are insensitive to TRAIL. In the present study, we show that oxaliplatin enhanced TRAIL‐induced apoptosis of MGC803, BGC823, and SGC7901 cells. Oxaliplatin promoted death receptor 4 (DR4) and death receptor 5 (DR5) clustering into aggregated lipid rafts, while the cholesterol‐sequestering agent nystatin partially prevented lipid raft aggregation, DR4 and DR5 clustering, and reduced apoptosis. Furthermore, the expression of the casitas B‐lineage lymphoma (Cbl) family was downregulated by oxaliplatin. Transfection of c‐Cbl or Cbl‐b partially reversed oxaliplatin‐induced lipid raft aggregation. These results indicated that oxaliplatin enhanced TRAIL‐induced gastric cancer cell apoptosis at least partially through Cbl‐regulated death receptor redistribution in lipid rafts.

β-Elemene Induces Apoptosis in Human Renal-cell Carcinoma 786-0 Cells through Inhibition of MAPK/ERK and PI3K/Akt/ mTOR Signalling Pathways

BACKGROUND Renal-cell carcinoma (RCC) is resistant to almost all chemotherapeutics and radiation therapy. β-Elemene, a promising anticancer drug extracted from a traditional Chinese medicine, has been shown to be effective against various tumors. In the present study, anti-tumor effects on RCC cells and the involved mechanisms were investigated. METHODS Human RCC 786-0 cells were treated with different concentrations of β-elemene, and cell viability and apoptosis were measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay and flow cytometry, respectively. Protein expression was assayed by western blotting. Autophagy was evaluated by transmission electron microscopy. RESULTS β-Elemene inhibited the viability of 786-0 cells in a dose- and time-dependent manner. The anti-tumor effect was associated with induction of apoptosis. Further study showed that β-elemene inhibited the MAPK/ERK as well as PI3K/Akt/mTOR signalling pathways. Moreover, robust autophagy was observed in cells treated with β-elemene. Combined treatment of β-elemene with autophagy inhibitors 3-methyladenine or chlorochine significantly enhanced the anti-tumor effects. CONCLUSIONS Our data provide first evidence that β-elemene can inhibit the proliferation of RCC 786- 0 cells by inducing apoptosis as well as protective autophagy. The anti-tumor effect was associated with the inhibition of MAPK/ERK and PI3K/Akt/mTOR signalling pathway. Inhibition of autophagy might be a useful way to enhance the anti-tumor effect of β -elemene on 786-0 cells.

Down-regulation of Cbl-b by bufalin results in up-regulation of DR4/DR5 and sensitization of TRAIL-induced apoptosis in breast cancer cells.

PurposeTNF-related apoptosis-inducing ligand (TRAIL) is a potential cancer therapeutic agent that preferentially induces apoptosis in cancer cells. However, breast cancer cells are generally resistant to TRAIL. Bufalin is a major active ingredient of the traditional Chinese medicine ChanSu. The present study aimed to assess the synergistic effect of bufalin and TRAIL and elucidate the underlying mechanisms in breast cancer cells.MethodsCell proliferation and apoptosis were measured by MTT assay and flow cytometry, respectively. The expression of proteins was assayed by flow cytometry and/or Western blotting. Transfection studies were used to determine the involvement of DR4, DR5 and Cbl-b in the synergistic effect of bufalin and TRAIL.ResultsMCF-7 and MDA-MB-231 cells were resistant to TRAIL. Both cell lines were dramatically sensitized to TRAIL-induced apoptosis by bufalin. Further experiments indicated that bufalin up-regulated DR4 and DR5, activated ERK, JNK and p38 MAPK and down-regulated Cbl-b. Blocking the up-regulation of DR4 and DR5 by siRNA rendered cells less sensitive to apoptosis induced by the combination of bufalin and TRAIL. Inhibition of the activation of ERK, JNK and p38 MAPK by specific inhibitors attenuated DR4 and DR5 up-regulation. Moreover, down-regulation of Cbl-b by shRNA led to stronger activation of ERK, JNK and p38 MAPK, more up-regulation of DR4 and DR5, and a stronger synergistic effect of bufalin and TRAIL.ConclusionsBufalin enhanced TRAIL-induced apoptosis by up-regulating the expression of DR4 and DR5. Bufalin-induced down-regulation of Cbl-b contributed to the up-regulation of DR4 and DR5, which might be partially mediated by the activation of ERK, JNK and p38 MAPK.

Reversal of P-glycoprotein-mediated multi-drug resistance by the E3 ubiquitin ligase Cbl-b in human gastric adenocarcinoma cells†

P‐glycoprotein (P‐gp)‐mediated multi‐drug resistance (MDR) is a major barrier to the effective chemotherapy of many cancers. Recent studies have shown that inhibition of the PI3K/Akt signalling pathway can reverse P‐gp‐mediated MDR. We investigated the expression of activated Akt (p‐Akt) in 124 human gastric carcinoma tissue samples. Ubiquitous p‐Akt expression was recorded in the majority (88/124). There was a significant correlation between p‐Akt expression and the expression of P‐gp. In the adriamycin‐resistant MDR gastric carcinoma cell line SGC7901/ADR, p‐Akt expression was increased in comparison with the parental cell line SGC7901. Treatment of SGC7901/ADR cells with the PI3K inhibitor LY294002 reduced the expression of both p‐Akt and P‐gp. To explore the role of ubiquitin ligase Cbl‐b in this regulatory pathway, SGC7901/ADR cells were transfected with a plasmid overexpressing wild‐type Cbl‐b. This down‐regulated the expression of both p‐Akt and P‐gp. Furthermore, resistance to chemotherapeutic drugs was partially reversed. These results demonstrate an important role for Cbl‐b in reversing P‐gp‐mediated gastric cancer MDR through suppression of the PI3K/Akt signalling pathway and the down‐regulation of P‐gp expression. Copyright

Ubiquitin ligase Cbl-b represses IGF-I-induced epithelial mesenchymal transition via ZEB2 and microRNA-200c regulation in gastric cancer cells

BackgroundInsulin-like growth factor I (IGF-I) can induce epithelial mesenchymal transition (EMT) in many epithelial tumors; however, the molecular mechanism by which this occurs is not clearly understood. Additionally, little is known about the involvement of IGF-I in gastric cancer.MethodsTwo gastric cancer cell lines were treated with IGF-I to induce EMT and levels of transcription factor ZEB2 and microRNA-200c (miR-200c) were measured. Cells were treated with Akt/ERK inhibitors to investigate the role of these pathways in IGF-I-mediated EMT. Transfection of shRNA plasmids was used to silence the ubiquitin ligase Cbl-b to assess its involvement in this process. The relationship between IGF-IR and Cbl-b expression, and the effect of IGF-IR and Cbl-b on metastasis were analyzed in primary gastric adenocarcinoma patients.ResultsIGF-I-induced gastric cancer cell EMT was accompanied by ZEB2 up-regulation. Furthermore, both Akt/ERK inhibitors and knockdown of Akt/ERK gene reversed IGF-I-induced ZEB2 up-regulation and EMT through up-regulation of miR-200c, suggesting the involvement of an Akt/ERK-miR-200c-ZEB2 axis in IGF-I-induced EMT. The ubiquitin ligase Cbl-b also ubiquitinated and degraded IGF-IR and inhibited the Akt/ERK-miR-200c-ZEB2 axis, leading to the repression of IGF-I-induced EMT. There was a significant negative correlation between the expression of IGF-IR and Cbl-b in gastric cancer patient tissues (r = -0.265, p < 0.05). More of patients with IGF-IR-positive expression and Cbl-b-negative expression were with lymph node metastasis (p < 0.001).ConclusionsTogether, these findings demonstrate that the ubiquitin ligase Cbl-b represses IGF-I-induced EMT, likely through targeting IGF-IR for degradation and further inhibiting the Akt/ERK-miR-200c-ZEB2 axis in gastric cancer cells.

The role of cbl family of ubiquitin ligases in gastric cancer exosome-induced apoptosis of Jurkat T cells.

Background. Exosomes are nanometer-sized vesicles with immunomodulatory functions, which are released by a diverse range of living cells. Although recent studies have shown that tumor-derived exosomes can suppress the function of T cells, the molecular mechanisms are not well understood. In the present study, we investigated the role of the Casitas B lineage lymphoma (cbl) family of ubiquitin ligases in gastric cancer exosome-induced apoptosis of Jurkat T cells. Materials and methods. By serial centrifugation and sucrose gradient ultracentrifugation, we isolated and purified the exosomes from gastric cancer SGC7901 cells, and identified them by electron microscopy and Western blotting. Cell apoptosis was detected using propidium iodide staining. Western blotting and RT-PCR was exploited to evaluate the expression of proteins and mRNA, respectively. Results. Gastric cancer exosomes induced Jurkat T cell apoptosis in a time- and dose-dependent manner and activated caspases 3, 8 and 9. The expression of Cbl-b and c-Cbl was up-regulated during exosome-induced apoptosis of cells. Meanwhile, exosomes induced ubiquitination of the p85 subunit of phosphoinositide 3-kinase (PI3K) and reduced downstream Akt activity. Inhibition of proteasome led to partial restoration of Akt activity and cell apoptosis. Discussion and Conclusions. The Cbl family of ubiquitin ligases might be involved in regulation of exosome-induced apoptosis of Jurkat T cells by increasing PI3K proteasome degradation, inactivation of PI3K/Akt signaling, thus mediating some effects of caspase activation.