Tumor immune microenvironment-based therapies in pancreatic ductal adenocarcinoma: time to update the concept.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal solid tumors. The tumor immune microenvironment (TIME) formed by interactions among cancer cells, immune cells, cancer-associated fibroblasts (CAF), and extracellular matrix (ECM) components drives PDAC in a more immunosuppressive direction: this is a major cause of therapy resistance and poor prognosis. In recent years, research has advanced our understanding of the signaling mechanism by which TIME components interact with the tumor and the evolution of immunophenotyping. Through revolutionary technologies such as single-cell sequencing, we have gone from simply classifying PDACs as "cold" and "hot" to a more comprehensive approach of immunophenotyping that considers all the cells and matrix components. This is key to improving the clinical efficacy of PDAC treatments. In this review, we elaborate on various TIME components in PDAC, the signaling mechanisms underlying their interactions, and the latest research into PDAC immunophenotyping. A deep understanding of these network interactions will contribute to the effective combination of TIME-based therapeutic approaches, such as immune checkpoint inhibitors (ICI), adoptive cell therapy, therapies targeting myeloid cells, CAF reprogramming, and stromal normalization. By selecting the appropriate integrated therapies based on precise immunophenotyping, significant advances in the future treatment of PDAC are possible.

Kang-Ai Injection Inhibits Gastric Cancer Cells Proliferation through IL-6/STAT3 Pathway.

OBJECTIVE: To explore the mechanisms underlying the proliferative inhibition of Chinese herbal medicine Kang-Ai injection (KAI) in gastric cancer cells. METHODS: Gastric cancer cell lines MGC803 and BGC823 were treated by 0, 0.3%, 1%, 3% and 10% KAI for 24, 48 and 72 h, respectively. The cell proliferation was evaluated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The apoptosis and cell cycle were evaluated by flow cytometry. Interleukin (IL)-6 mRNA and protein expression levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immune sorbent assay (ELISA), respectively. The protein expression levels of cyclin A, cyclin E, cyclin B1, cyclin D1, p21, retinoblastoma (RB), protein kinase B (AKT), extracellular regulated protein kinases (ERK), signal transducer and activator of transcription (STAT) 1 and STAT3 were detected by Western blot. RESULTS: KAI inhibited the proliferation of MGC803 and BGC823 gastric cancer cells in dose- and time-dependent manner. After treated with KAI for 48 h, the proportion of G1 phase was increased, expression level of cyclin D1 and phosphorylation-RB were down-regulated, whereas the expression of p21 was up-regulated (all P<0.01). Furthermore, 48-h treatment with KAI decreased the phosphorylation level of STAT3, inhibited the mRNA and protein expressions of IL-6 (all P<0.01). IL-6 at dose of 10 ng/mL significantly attenuated the proliferative effect of both 3% and 10% KAI, and recovered KAI-inhibited STAT3 phosphorylation and cyclin D1 expression level (all P<0.01). CONCLUSION: KAI exerted an anti-proliferative function by inhibiting IL-6/STAT3 signaling pathway followed by the induction of G1 phase arrest in gastric cancer cells.

Bufalin inhibits peritoneal dissemination of gastric cancer through endothelial nitric oxide synthase-mitogen-activated protein kinases signaling pathway.

Peritoneal dissemination threatens the survival of patients with gastric cancer (GC). Bufalin is an extract of traditional Chinese medicine, which has been proved to have anticancer effect. The target of bufalin in suppressing gastric cancer peritoneal dissemination (GCPD) and the underlying mechanism are still unclear. In this research, GC cell line MGC-803 and high-potential peritoneal dissemination cell line MKN-45P were treated with bufalin or L-NAME. Malignant biological behavior and protein level of GC cell lines were detected with MTT, wound healing, transwell, adhesion, and western blotting. Bioinformatics analysis and patient tissues were used to verify the role of endothelial nitric oxide synthase (NOS3) in GC. Mice model was used to assess the effect of bufalin and role of NOS3 in vivo. We found that bufalin inhibited the proliferation, invasion, and migration in GC cell lines. NOS3, which was an independent prognostic factor of GC patients, was predicted to be a potential target of bufalin. Further experiments proved that bufalin reduced the phosphorylation of NOS3, thereby inhibiting the mitogen-activated protein kinase (MAPK) signaling pathway, and ultimately suppressed GCPD by inhibiting EMT process. In conclusion, NOS3 was a potential therapeutic target and prognostic biomarker of GC. Bufalin could suppress GCPD through NOS3-MAPK signaling pathway, which provided more evidence support for intraperitoneal perfusion of bufalin to treat GCPD.

ß-Elemene inhibits the metastasis of multidrug-resistant gastric cancer cells through miR-1323/Cbl-b/EGFR pathway.

BACKGROUND: ß-Elemene is a natural agent extracted from the traditional Chinese herbal medicine Curcuma wenyujin that is a promising novel plant-derived drug with broad-spectrum anticancer activity. Our previous study identified an enhanced capacity for metastasis in multidrug resistant (MDR) gastric cancer and breast cancer cells. However, the anti-metastatic effects of ß-Elemene on MDR cancer cells remain unknown. PURPOSE: In this study, we posit the hypothesis that ß-elemene possesses antimetastatic effects on MDR cancer cells. METHODS: Cell viability assay was used to assess the resistance of SGC7901/ADR cells and the cytotoxic effects of ß-Elemene. Wound healing, transwell assay and lung metastatic mice model were used to the anti-metastasis effects of ß-Elemene. MicroRNA microarray analysis was used to explore potential regulated miRNAs. Luciferase reporter assay was used to identify the direct target. Human MMP antibody array, western blot, immunoprecipitation, qRT-PCR analyses and immunohistochemistry were conducted to investigate the underlying anti-metastasis mechanism of ß-Elemene. RESULTS: In this study, we found that ß-Elemene significantly inhibited the metastatic capacity of MDR gastric cells in vivo and in vitro. Mechanistically, we found that ß-Elemene regulated MMP-2/9 expression and reversed epithelial-mesenchymal transition. Further studies showed that ß-Elemene upregulated Cbl-b expression, resulting in inhibition of the EGFR-ERK/AKT pathways, which regulate MMP-2/9. Additionally, we confirmed that ß-Elemene upregulated Cbl-b by inhibiting miR-1323 expression. Finally, we found that numbers of metastatic tumor nodules were significantly decreased in the lungs of nude mice after ß-Elemene treatment. CONCLUSION: Our results suggested that ß-Elemene inhibits the metastasis of MDR gastric cancer cells by modulating the miR-1323/Cbl-b/EGFR signaling axis.

ß-Elemene inhibits peritoneal metastasis of gastric cancer cells by modulating FAK/Claudin-1 signaling.

Peritoneal metastasis is common in advanced gastric cancer patients and is typically associated with a worse prognosis. ß-Elemene is a natural compound that can be isolated from the Curcuma wenyujin plant and has been widely used in China to treat a variety of cancers. However, the anti-metastatic impacts of ß-elemene on gastric cancer remain unknown. In our study, we found that ß-elemene significantly inhibited the migration and invasive capacity of gastric cells in vitro and inhibited the capacity of gastric cancer cells to peritoneally diffuse and metastasize in vivo. Mechanistically, we demonstrated that the anti-metastatic effects of ß-elemene were exerted by downregulating the expression of Claudin-1. Furthermore, ß-elemene was found to inhibit the metastatic capacity of cells by downregulating FAK phosphorylation, which regulated Claudin-1. Overall, our result revealed that ß-elemene inhibited peritoneal metastases from gastric cancer by modulating the FAK/Claudin-1 pathway.

Bufalin induces protective autophagy by Cbl-b regulating mTOR and ERK signaling pathways in gastric cancer cells.

Bufalin, a natural small-molecule compound derived from the traditional Chinese medicine Chan su, has shown promising anti-cancer effects against a broad variety of cancer cells through different mechanisms. It has been reported to induce autophagy in gastric cancer cells. However, the molecular mechanism involved is not fully elucidated. In the present study, we aimed to investigate the molecular mechanism by which bufalin induce autophagy in human gastric cancer cells. We found that bufalin induced apoptosis and autophagy in gastric cancer cells, and autophagy prevented human gastric cancer cells from undergoing apoptosis. Bufalin treatment changed the expression of autophagy-related proteins. Moreover, phosphorylated Akt, mTOR, and p70S6K were all significantly decreased, while phosphorylated ERK1/2 was increased by bufalin. Pretreatment of MGC803 cells with the ERK1/2-specific inhibitor PD98059 led to the down-regulation of LC3 II. Further study showed that Cbl-b positively regulated autophagy by suppressing mTOR and enhancing ERK1/2 activation. Therefore, our data provide evidence that bufalin induces autophagy in MGC803 cells via both Akt/mTOR/p70S6K and ERK signaling pathways, and Cbl-b-mediated suppression of mTOR and activation of ERK1/2 might play an important role.

ß-elemene increases the sensitivity of gastric cancer cells to TRAIL by promoting the formation of DISC in lipid rafts.

ß-Elemene, an anti-cancer drug extracted from traditional Chinese medicinal herb, showed anti-tumor effects on gastric cancer cells. Our previous studies reported gastric cancer cells are insensitive to TRAIL. However, whether ß-elemene could enhance anti-cancer effects of TRAIL on gastric cancer cells is unknown. In our present study, ß-elemene prevented gastric cancer cell viability in dose-dependent manner, and when combined with TRAIL, obviously inhibited proliferation and promoted apoptosis in gastric cancer cells. Compared to ß-elemene or TRAIL alone, treatment with ß-elemene and TRAIL obviously promoted DR5 clustering as well as translocation of Caspase-8, DR5 and FADD into lipid rafts. This led to cleavage of Caspase-8 and the formation of death-inducing signaling complex (DISC) in lipid rafts. The cholesterol-sequestering agent nystatin partially reversed DR5 clustering and DISC formation, preventing apoptosis triggered by the combination of ß-elemene and TRAIL. Our results suggest that ß-elemene increases the sensitivity of gastric cancer cells to TRAIL partially by promoting the formation of DISC in lipid rafts.

Bufalin inhibits TGF-ß-induced epithelial-to-mesenchymal transition and migration in human lung cancer A549 cells by downregulating TGF-ß receptors.

The epithelial-to-mesenchymal transition (EMT) is a well-known prerequisite for cancer cells to acquire the migratory and invasive capacity, and to subsequently metastasize. Bufalin is one of the major active components of the traditional Chinese medicine Chan Su, and accumulating evidence has shown its anticancer effect in multipe types of cancer. However, the role of bufalin in transforming growth factor­ß (TGF­ß)­induced EMT and migration remains unclear. In the present study, the effect of bufalin on TGF­ß­induced EMT and migration was investigated in human lung cancer A549 cells. TGF­ß induced EMT in A549 cells and increased their migratory ability, which were markedly suppressed by bufalin. Additionally, TGF­ß­induced upregulation of Twist2 and zinc finger E­box binding homeobox 2 (ZEB2), as well as the phosphorylation of Smad2 and Smad3 were also inhibited by bufalin. However, the Smad­independent signaling pathways were not affected. Further analysis showed that the TGF­ß receptor I (TßRI) and TGF­ß receptor II (TßRII) were downregulated in the presence of bufalin. Pretreatment with SB431542, a potent inhibitor of the phosphorylation of TßRI, significantly attenuated TGF­ß­induced EMT, mimicking the effect of bufalin on A549 cells. Taken together, these results suggest that bufalin suppresses TGF-ß-induced EMT and migration by downregulating TßRI and TßRII in A549 cells.

Early initiation of fluorouracil-based adjuvant chemotherapy improves survival in patients with resectable gastric cancer.

PURPOSE: Several clinical trials have suggested that adjuvant chemotherapy improves the survival of patients with resected gastric cancer, but the optimal time at which to initiate post-operative adjuvant chemotherapy has not been studied. This study investigated the association between time to adjuvant chemotherapy and survival in gastric cancer. METHODS: We retrospectively identified 266 patients with stage IB-IIIC gastric cancer who received fluorouracil-based adjuvant chemotherapy after radical gastrectomy. Overall survival (OS) was compared between patients grouped according to time from surgery to adjuvant chemotherapy (<45 and ≥45 days). The Cox proportional hazards model was used to analyze the effects of time to initiation of chemotherapy and other clinical covariates on survival. RESULTS: Of 266 patients, 141 (53%) started adjuvant chemotherapy within 45 days after surgery and 125 (47%) started adjuvant chemotherapy more than 45 days after surgery. The 3-year OS rates were 81.2 and 65.8% for patients starting chemotherapy within 45 days and after 45 days, respectively (p=0.006). Multivariate analysis identified early initiation of adjuvant chemotherapy, completion of the planned chemotherapy, and early-stage disease as favorable prognostic factors in terms of OS (p<0.05). Subgroup analysis suggested that starting chemotherapy within 45 days after surgery was associated with significant OS benefit compared with initiation of chemotherapy after 45 days from surgery in most subgroups. CONCLUSIONS: This retrospective analysis suggests that delaying adjuvant chemotherapy for longer than 45 days after surgery may be associated with poorer survival in patients with resected gastric cancer.

Microsatellite instability and survival in gastric cancer: A systematic review and meta-analysis.

Microsatellite instability (MSI) is associated with the prognosis in several cancers and is used for determination of the chemotherapy regimen in stage II colon cancer in the National Comprehensive Cancer Network guideline. However, the association between MSI and the prognosis of gastric cancer remains unclear. PubMed database was searched until January 2014 using MeSH terms and key words to identify the studies evaluating MSI and prognosis of gastric cancer and the references were manually searched. The main outcome was the overall survival rate and the subordinate outcome was the association between high-frequency MSI (MSI-H) and clinicopathological characteristics. Eight studies met the inclusion criteria and the majority of data were collected retrospectively. There were 1,976 patients, 431 of which were MSI-H patients, with a range of 11.68-33.82%. Four studies used the National Cancer Institute panel to define MSI-H, the other four had microsatellite markers ranging 2-11. Significant associations were found in three studies and the overall summary estimate was hazard ratio, 0.63 (95% confidence interval, 0.52-0.77), with no evidence of inter-study heterogeneity (I2=0.0%). MSI-H patients were identified to have a tendency to have less lymph node (LN) metastasis, superficial tumor invasion and to be intestinal type. In conclusion, MSI-H gastric cancers have an improved prognosis, accompanied with reduced risk of LN metastasis, tumor invasion and mortality.

Secreted protein acidic and rich in cysteine antagonizes bufalin-induced apoptosis in gastric cancer cells.

Bufalin is an active compound in the traditional Chinese medicine Chan Su, which has been shown to induce apoptosis in a range of cancer cell types. However, certain gastric cancer cells are known to be resistant to bufalin. Intracellular secreted protein acidic and rich in cysteine (SPARC) regulates proliferation and apoptosis. This study aimed to evaluate the role of SPARC in bufalin-induced apoptosis in SGC7901 and MGC803 gastric cancer cells. SGC7901 cells with high SPARC expression were more resistant to bufalin than MGC803 cells with low SPARC expression. This resistance was significantly reversed by small interfering (si)RNA-mediated knockdown of SPARC. Furthermore, it was shown that SPARC negatively regulated bufalin-induced intrinsic apoptosis by protecting mitochondrial integrity, decreasing the release of cytoplasmic cytochrome c and increasing the ratio of Bcl-2/Bax. In addition, SPARC overcame bufalin-induced G2/M phase arrest by increasing levels of Cyclin B1 and Cyclin A protein expression. SPARC also activated cellular survival signals, including Src and Akt, but not extracellular signal-regulated kinase. This study demonstrated that SPARC antagonizes bufalin-induced apoptosis via inhibition of the intrinsic apoptosis pathway, inhibition of cell cycle arrest and activation of certain pathways involved in proliferation. This provides novel evidence for SPARC as a potential target by which to sensitize gastric cancer cells to bufalin.

The role of E3 ubiquitin ligase Cbl proteins in ß-elemene reversing multi-drug resistance of human gastric adenocarcinoma cells.

Recent studies indicate that ß-elemene, a compound isolated from the Chinese herbal medicine Curcuma wenyujin, is capable of reversing tumor MDR, although the mechanism remains elusive. In this study, ß-Elemene treatment markedly increased the intracellular accumulation of doxorubicin (DOX) and rhodamine 123 in both K562/DNR and SGC7901/ADR cells and significantly inhibited the expression of P-gp. Treatment of SGC7901/ADR cells with ß-elemene led to downregulation of Akt phosphorylation and significant upregulation of the E3 ubiquitin ligases, c-Cbl and Cbl-b. Importantly, ß-elemene significantly enhanced the anti-tumor activity of DOX in nude mice bearing SGC7901/ADR xenografts. Taken together, our results suggest that ß-elemene may target P-gp-overexpressing leukemia and gastric cancer cells to enhance the efficacy of DOX treatment.

Optimal duration of fluorouracil-based adjuvant chemotherapy for patients with resectable gastric cancer.

BACKGROUND: Although several clinical trials have suggested that postoperative adjuvant chemotherapy can improve survival of patients with gastric cancer, the optimal treatment duration has not been studied. This retrospective analysis evaluated the outcomes of patients with gastric cancer treated with six cycles of fluorouracil-based treatment compared with a cohort treated with four or eight cycles. METHODS: We retrospectively identified 237 patients with stage IB-IIIC gastric cancer who received four, six, or eight cycles of fluorouracil-based adjuvant chemotherapy administered every 3 weeks after radical gastrectomy. The endpoint was overall survival (OS). Factors associated with prognosis were also analyzed. RESULTS: The estimated 3-year OS rates for the four-, six-, and eight-cycle cohorts were 54.4%, 76.1%, and 68.9%, respectively; and the estimated 5-year OS rates were 41.2%, 74.0%, and 65.8%, respectively. Patients who received six cycles were more likely to have a better OS than those who received four cycles (P = 0.002). Eight cycles failed to show an additional survival benefit (P = 0.454). In the multivariate analysis, the number of chemotherapy cycles was associated with OS independent of clinical covariates (P<0.05). Subgroup analysis suggested that among patients in all age groups examined, male patients, and subgroups of fluorouracil plus oxaliplatin combined chemotherapy, stage III, poor differentiation, and gastrectomy with D2 lymphadenectomy, six cycles of adjuvant chemotherapy were associated with a statistically significant benefit of OS compared with four cycles (P<0.05). CONCLUSIONS: Six cycles of adjuvant chemotherapy might lead to a favorable outcome for patients with gastric cancer, and two further cycles could not provide an additional clinical benefit.

ß-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. ß-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 ß-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: ß-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 ß-elemene inhibited the MAPK/ERK as well as PI3K/Akt/mTOR signalling pathways. Moreover, robust autophagy was observed in cells treated with ß-elemene. Combined treatment of ß-elemene with autophagy inhibitors 3-methyladenine or chlorochine significantly enhanced the anti-tumor effects. CONCLUSIONS: Our data provide first evidence that ß-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 ß -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.

PURPOSE: TNF-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. METHODS: Cell 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. RESULTS: MCF-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. CONCLUSIONS: Bufalin 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.

ß-Elemene induces apoptosis as well as protective autophagy in human non-small-cell lung cancer A549 cells.

OBJECTIVES: ß-Elemene, a novel traditional Chinese medicine, has been shown to be effective against a wide range of tumours. In this study, the antitumour effect of ß-elemene on human non-small-cell lung cancer (NSCLC) A549 cells and the mechanism involved have been investigated. METHODS: Cell viability and apoptosis were measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry, respectively. Protein expression was assayed by Western blotting. Autophagy was evaluated under fluorescence microscopy and transmission electron microscopy. KEY FINDINGS: ß-Elemene inhibited the viability of A549 cells in a dose-dependent manner. This suppression of cell viability was due to the induction of apoptosis. Further study showed that ß-elemene inhibited the activity of the PI3K/Akt/mTOR/p70S6K1 signalling pathway, and at the same time it triggered a robust autophagy. The autophagy was characterized by the accumulation of punctate LC3 dots in the cytoplasm, morphological changes, and the increased levels of LC3-II as well as Atg5-Atg12 conjugated proteins. Inhibition of autophagy with chlorochine significantly enhanced the antitumour effect of ß-elemene. CONCLUSIONS: Our data indicated that ß-elemene inhibited the activity of the PI3K/Akt/mTOR/p70S6K1 signalling pathway in human NSCLC A549 cells, which resulted in apoptosis as well as protective autophagy. A combination of ß-elemene with autophagy inhibitor might be an effective therapeutic option for advanced NSCLC.

Synergistic antitumor effect of ß-elemene and etoposide is mediated via induction of cell apoptosis and cell cycle arrest in non-small cell lung carcinoma cells.

ß-Elemene, an anticancer agent, was isolated from the traditional Chinese medicine plant, curcuma aromatica. In this study, we investigated the synergistic antitumor effect of ß-elemene and etoposide phosphate (VP-16) in A549 non-small cell lung carcinoma cells. The cells were treated with ß-elemene (20 or 50 µg/ml), VP-16 (15 µg/ml) or the combination of both for 24 h. Compared to the treatment with ß-elemene or VP-16 alone, an increased antitumor activity was observed with the combination of both, which was mediated by the cleavage of PARP, the up-regulation of Bax, p53 and p21, and the suppression of cyclin D1. These results suggest that the combination of ß-elemene and VP-16 may be a promising therapeutic option for lung cancer.

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

BACKGROUND: ß-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 ß-elemene kills cells remains unclear. The aim of the present study is to investigate the anti-tumor effect of ß-elemene on human gastric cancer cells and the molecular mechanism involved. RESULTS: ß-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 ß-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 ß-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 ß-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 ß-elemene. CONCLUSIONS: Our data provides the first evidence that ß-elemene induces protective autophagy and prevents human gastric cancer cells from undergoing apoptosis. A combination of ß-elemene with autophagy inhibitor might thus be a useful therapeutic option for advanced gastric cancer.

[Effect of bufalin-inducing apoptosis on Bcl-2 and PKC in HL-60 cells].

Previous study revealed that bufalin can inhibit proliferation, and induce apoptosis in some human cancer cell lines. However, the mechanism of its anticancer effect has not been fully understood. The present study was designed to investigate the effects of bufalin-induced apoptosis on Bcl-2 and PKC in human leukemic HL-60 cells. The cell viability was determined by trypan blue dye exclusion. The apoptosis was detected by morphology, flow cytometry and DNA agarose gel electrophoresis. The expressions of Bcl-2 and PKC were analyzed by Western blot, and activity of PKC was assayed by [gamma-(32)P] isotope incorporation method. The results showed as follows: (1) proliferation of HL-60 cells was inhibited by bufalin and the IC(50) at 24, 48, 72 hours were (25.8 +/- 2.1), (8.0 +/- 1.2) and (2.3 +/- 0.3) nmol/L, respectively. (2) apoptosis of HL-60 cells was induced when the cells were treated with bufalin at concentration of 50 nmol/L for 24 hours. (3) compared with control, treatment with bufalin at concentration of 50 nmol/L for 6 - 24 hours resulted in downregulation of protein expression, decrease of phosphorylation, and cleavage of Bcl-2, simultaneously. (4) the activity of total PKC was unchanged when HL-60 cells were exposed to 1 - 100 nmol/L bufalin for 30 minutes, but PKCbetaII underwent translocation from cytosol to membrane. It is concluded that apoptosis induced by bufalin is associated with downregulation of protein expression, dephosphorylation, and cleavage of Bcl-2 in HL-60 cells.

Negative regulation of FcepsilonRI-mediated mast cell activation by a ubiquitin-protein ligase Cbl-b.

Aggregation of the high-affinity immunoglobulin E (IgE) receptor (FcepsilonRI) on mast cells induces a number of biochemical events, including protein-tyrosine phosphorylation leading to degranulation and multiple cytokine gene transcription. Here, we have demonstrated that a second member of the Cbl family of ubiquitin-protein ligase Cbl-b translocates into the lipid raft after FcepsilonRI engagement. Overexpression of Cbl-b in the lipid raft inhibits FcepsilonRI-mediated degranulation and cytokine gene transcription through the distinct mechanism. A point mutation of Cys373 in the RING finger domain of Cbl-b abrogates the suppression of FcepsilonRI-mediated degranulation but not cytokine gene transcription. The antigen-induced tyrosine phosphorylation of FcepsilonRI, Syk, phospholipase C-gamma (PLC-gamma), activation of c-Jun N-terminal kinase (JNK), extracellular signal regulated kinase (ERK), inhibitor of nuclear factor kappaB kinase (IKK), and Ca++ influx were all suppressed in the cells overexpressing Cbl-b in the lipid raft. In particular, the expression amount of Gab2 protein and thereby its FcepsilonRI-mediated tyrosine phosphorylation were dramatically down-regulated by ubiquitin-protein ligase activity of Cbl-b. These results suggest that Cbl-b is a negative regulator of both Lyn-Syk-LAT and Gab2mediated complementary signaling pathways in FcepsilonRI-mediated mast cell activation.