Oridonin enhances the anti-tumor activity of gemcitabine towards pancreatic cancer by stimulating Bax- and Smac-dependent apoptosis.

BACKGROUND: Oridonin has been shown to exhibit potent anti-tumor activity, but the exact mechanisms underlying this activity remains unclear. Here, we investigated whether oridonin could synergistically enhance the activity of gemcitabine against BxPC-3 pancreatic cancer cells. METHODS: CCK-8 assays were conducted to determine cell viability. The cellular morphology was observed under light microscope and compared with normal cell. Apoptotic cells were quantified by flow cytometry. RT-PCR, Western blot analysis and immunohistochemical methods were employed to analyze related-gene and protein expression. A xenograft tumor model was conducted whereby BxPC-3 cells were introduced into nude mice to detect anti-tumor effects in vivo. RESULTS: In vitro, oridonin inhibited the proliferation of BxPC-3 and Panc-1 cells cells in a concentration and time dependent manner. In addition, oridonin dose-dependently induced Panc-1 cellular morphology changes. Besides, In BxPC-3 cells oridonin potentiated gemcitabine-induced apoptosis. oridonin induced Bax translocation from cytosolic to mitochondrial compartments. This was accompanied by the release of the apoptogenic protein Smac and inhibition of its downstream target XIAP. These effects were further enhanced by combined treatment with oridonin and gemcitabine. In vivo, both oridonin alone and in combination with gemcitabine significantly suppressed tumor growth in a Bax- and Smac-dependent manner. CONCLUSIONS: Oridonin can potentiate the effects of gemcitabine through Bax- and Smac-dependent mitochondrial signaling pathways in BxPC-3 pancreatic cancer cells. Therefore, oridonin has the potential to be used clinically in the treatment of pancreatic cancer.

The inhibitory effect of oridonin on colon cancer was mediated by deactivation of TGF-ß1/Smads-PAI-1 signaling pathway in vitro and vivo.

BACKGROUND: Oridonin, the main active component of Rabdosia rubescens, has been demonstrated to have anti-tumor effect on all kinds of cancer cells through various mechanisms and it has shown antitumor activity in some tumors partially via the suppression of TGF-ß/Smads signaling pathway. The aim of this study was to explore the anticancer effect of oridonin on human colon carcinoma and underlying mechanism in vitro and vivo. METHODS: CCK-8 assay was employed to assess cell viability. The key target genes and proteins involved in TGF-ß/Smads pathway was detected by RT-PCR, Western blotting and immunohistochemistry. The orthotopic transplantation tumor model of colon cance LOVO cell was introduced to detect anti-cancer effects in vivo. RESULTS: Oridonin inhibited the proliferation of colon cancer LOVO cells in a concentration and time dependent manner. In addition, oridonin reduced the levels of Smad2, Smad3, Smad4, PAI-1 and the phosphorylation of Smad2 and Smad3 induced by TGF-ß1 in vitro. Subsequently, we established an orthotopically implanted tumor model in nude mice and found that oridonin treatment significantly suppressed tumor growth, and which was accompanied by the down-regulation of Smad2, Smad3, Smad4, PAI-1 and p-Smad2, p-Smad3 expression levels. CONCLUSION: Our present study demonstrated that the growth inhibition of colon cancer by oridonin could be partially mediated through discontinuing TGF-ß1/Smads-PAI-1 signaling pathway, suggesting it as a promising agent in treating colorectal cancer.

Emodin enhances the demethylation by 5-Aza-CdR of pancreatic cancer cell tumor-suppressor genes P16, RASSF1A and ppENK.

5-Aza-2'-deoxycytidine (5-Aza-CdR) is currently acknowledged as a demethylation drug, and causes a certain degree of demethylation in a variety of cancer cells, including pancreatic cancer cells. Emodin, a traditional Chinese medicine (TCM), is an effective monomer extracted from rhubarb and has been reported to exhibit antitumor activity in different manners in pancreatic cancer. In the present study, we examined whether emodin caused demethylation and increased the demethylation of three tumor-suppressor genes P16, RASSF1A and ppENK with a high degree of methylation in pancreatic cancer when combined with 5-Aza-CdR. Our research showed that emodin inhibited the growth of pancreatic cancer Panc-1 cells in a dose- and time-dependent manner. Dot-blot results showed that emodin combined with 5-Aza-CdR significantly suppressed the expression of genome 5mC in PANC-1 cells. In order to verify the effect of methylation, methylation-specific PCR (MSP) and bisulfite genomic sequencing PCR (BSP) combined with TA were selected for the cloning and sequencing. Results of MSP and BSP confirmed that emodin caused faint demethylation, and 5-Aza-CdR had a certain degree of demethylation. When emodin was combined with 5-Aza-CdR, the demethylation was more significant. At the same time, fluorescent quantitative PCR and western blot analysis results confirmed that when emodin was combined with 5-Aza-CdR, the expression levels of P16, RASSF1A and ppENK were increased more significantly compared to either treatment alone. In contrast, the expression levels of DNA methyltransferase 1 (DNMT1) and DNMT3a were more significantly reduced with the combination treatment than the control or either agent alone, further proving that emodin in combination with 5-Aza-CdR enhanced the demethylation effect of 5-Aza-CdR by reducing the expression of methyltransferases. In conclusion, the present study confirmed that emodin in combination with 5-Aza-CdR enhanced the demethylation by 5-Aza-CdR of tumor-suppressor genes p16, RASSF1A and ppENK by reducing the expression of methyltransferases DNMT1 and DNMT3a.

Effects of emodin on the demethylation of tumor-suppressor genes in pancreatic cancer PANC-1 cells.

Emodin, a natural anthraquinone derivative isolated from Rheum palmatum, has been reported to inhibit the growth of pancreatic cancer cells through different modes of action; yet, the detailed mechanism remains unclear. In the present study, we hypothesized that emodin exerts its antitumor effect by participating in the regulation of the DNA methylation level. Our research showed that emodin inhibited the growth of pancreatic cancer PANC-1 cells in a dose- and time-dependent manner. Dot-blot results showed that 40 µM emodin significantly inhibited genomic 5 mC expression in the PANC-1 cells, and mRNA-Seq showed that different concentrations of emodin could alter the gene expression profile in the PANC-1 cells. BSP confirmed that the methylation levels of P16, RASSF1A and ppENK were decreased, while concomitantly the unmethylated status was increased. RT-PCR and western blotting results confirmed that the low expression or absence of expression of mRNA and protein in the PANC-1 cells was re-expressed following treatment with emodin. In conclusion, our study for the first time suggests that emodin inhibits pancreatic cancer cell growth, which may be related to the demethylation of tumor-suppressor genes. The related mechanism may be through the inhibition of methyltransferase expression.

Enhancement of the effects of gemcitabine against pancreatic cancer by oridonin via the mitochondrial caspase-dependent signaling pathway.

Gemcitabine is a first­line chemotherapeutic agent used in the treatment of pancreatic cancer; however resistance of the disease to the drug often develops over time. Agents that can either enhance the effects of gemcitabine, or help to overcome the chemoresistance to the drug are needed for the successful treatment of pancreatic cancer. Oridonin is one such agent which is safe and multi­targeted and has previously been shown to induce apoptosis in other tumor cells, through mitochondrial signaling pathways. The aims of the present study were to evaluate whether oridonin may enhance the effects of gemcitabine on pancreatic cancer in vitro and to investigate the possible mechanisms of this enhancement. In vitro studies have previously shown that oridonin can inhibit the proliferation of the Panc­1 pancreatic cancer cell line, and potentiate gemcitabine­induced apoptosis, which was shown to be associated with cell cycle arrest in the G1 phase. Western blot and quantitative polymerase chain reaction analyses demonstrated that the expression levels of the anti­apoptotic gene Bcl­2 and the Bcl­2/Bax ratio in the oridonin and the oridonin plus gemcitabine groups were significantly downregulated as compared with the gemcitabine treatment and control groups. The expression levels of pro­apoptotic genes Bax, cytochrome c (cyt c), and caspase­3 and ­9 in the oridonin and the combination groups were significantly upregulated as compared with the other two groups. The results suggested that oridonin improved the anti­tumor effects of gemcitabine through the enhancement of gemcitabine­induced apoptosis.This mechanism may be through the downregulation of Bcl­2 expression and the upregulation of Bax expression, resulting in the reduction of the Bcl­2/Bax ratio. These effects may promote the release of cyt c from the mitochondria into the cytoplasm thus triggering the mitochondrial apoptosis signaling pathway. Furthermore, caspase­3 and ­9 were shown to be activated as a result of the induction of apoptosis.

Oridonin induces apoptosis in SW1990 pancreatic cancer cells via p53- and caspase-dependent induction of p38 MAPK.

Oridonin, an active component isolated from Rabdosia rubescens, has been reported to exhibit antitumor effects. In the present study, we evaluated the antitumor activity and the mechanisms of action of oridonin in pancreatic cancer. Oridonin treatment significantly induced apoptotic cell death in SW1990 pancreatic cancer cells in a dose-dependent manner. Additionally, cell apoptosis was markedly inhibited by PFT α (pifithrin α), a p53-specific inhibitor, which was applied to evaluate the function of p53, showing that p53 was responsible for the cytotoxity of oridonin. Moreover, oridonin increased the expression of p-p53 with a concomitant increase in p21 in the SW1990 cells. Following treatment with mitogen-activated protein kinase (MAPK) inhibitors, PD98059 (ERK inhibitor), SP600125 (JNK inhibitor) and SB203580 (p38 inhibitor), the cytotoxity of oridonin was not influenced by JNK (SP600125) and ERK (PD98059), but these effects were opposite to the cytotoxity of oridonin observed with SP203580 treatment. These findings confirmed that orodonin-induced apoptosis was p38-dependent, but JNK- and ERK-independent. Furthermore, the activation of the p38 kinase promoted the activation of p53 and its downstream target p21, and further caused caspase-9 and -3 activation, as demonstrated by evidence showing that the p38 inhibitor SB203580 not only blocked the phosphorylation of p38 but also reduced the activation of p53, p21 and caspase-9 and -3. Collectively, these results suggest that p53-dependent and caspase-dependent induction of p38 MAPK directly participates in apoptosis induced by oridonin.

Involvement of the phosphoinositide 3-kinase/Akt pathway in apoptosis induced by capsaicin in the human pancreatic cancer cell line PANC-1.

Capsaicin, one of the major pungent ingredients found in red peppers, has been recently demonstrated to induce apoptosis in various malignant cell lines through an unclear mechanism. In this study, the effect of capsaicin on proliferation and apoptosis in the human pancreatic cancer cell line PANC-1 and its possible mechanism(s) of action were investigated. The results of a Cell Counting Kit-8 (CCK-8) assay revealed that capsaicin significantly decreased the viability of PANC-1 cells in a dose-dependent manner. Capsaicin induced G0/G1 phase cell cycle arrest and apoptosis in PANC-1 cells as demonstrated by a flow cytometric assessment. Caspase-3 expression at both the protein and mRNA level was promoted following capsaicin treatment. Furthermore, we revealed that phospho-PI3 Kinase p85 (Tyr458) and phospho-Akt (Ser473) in PANC-1 cells were downregulated in response to capsaicin. Moreover, capsaicin gavage significantly inhibited the growth of pancreatic cancer PANC-1 cell xenografts in athymic nude mice. An increased number of TUNEL-positive cells and cleaved caspase-3 were observed in capsaicin-treated mice. In vivo, capsaicin downregulated the expression of phospho-PI3 Kinase p85 (Tyr458) and phospho-Akt (Ser473). In conclusion, we have demonstrated that capsaicin is an inhibitor of growth of PANC-1 cells, and downregulation of the phosphoinositide 3-kinase/Akt pathway may be involved in capsaicin-induced apoptosis in vitro and in vivo.

Oridonin enhances antitumor activity of gemcitabine in pancreatic cancer through MAPK-p38 signaling pathway.

Gemcitabine is currently the best treatment available for pancreatic cancer (PaCa); however, patients with the disease develop resistance to the drug over time. Agents that can either enhance the effects of gemcitabine or overcome chemoresistance to the drug are required for the treatment of PaCa. Oridonin is one such agent which is safe and multitargeted, and has been linked with the suppression of survival, proliferation, invasion and angiogenesis of cancer. In this study, we investigated whether oridonin could sensitize PaCa to gemcitabine in vitro and in vivo. In vitro, oridonin inhibited the proliferation of the PaCa cell line, BxPC-3, potentiated the apoptosis induced by gemcitabine, induced G1 cell cycle arrest and activated p38 and p53; these results were significant when oridonin was combined with gemcitabine. In vivo, we found that oridonin significantly suppressed tumor growth and this effect was further enhanced by gemcitabine (P<0.05). Tumors from nude mice injected with BxPC-3 PaCa cells and treated with a combination of oridonin and gemcitabine showed a significant upregulation in p38 and p53 activation (P<0.05 vs. control, P<0.05 vs. gemcitabine or oridonin alone). Taken together, our results demonstrate that oridonin can potentiate the effects of gemcitabine in PaCa through the mitogen-activated protein kinase (MAPK)-p38 signaling pathway, which is dependent on p53 activation.

Emodin potentiates the antitumor effects of gemcitabine in PANC-1 pancreatic cancer xenograft model in vivo via inhibition of inhibitors of apoptosis.

Pancreatic cancer is a highly aggressive malignant disease. Gemcitabine is currently the standard first-line chemotherapeutic agent for pancreatic cancer. As members of apoptosis inhibitors, Survivin and XIAP play an important role in chemotherapy resistance in pancreatic cancer. Emodin has therapeutic potential against cancers. This study was designed to investigate whether combination therapy with gemcitabine and emodin enhanced antitumor efficacy in pancreatic cancer. The application of the combination therapy triggered significantly higher frequency of pancreatic cancer cell apoptosis. Our research demonstrated that the combination of emodin and gemcitabine resulted in significantly reduced tumor volumes compared to gemcitabine or emodin treatment alone. Immunohistochemistry and western immunoblot analyses showed that Survivin and XIAP expression were downregulated in emodin and the combination groups compared to the other two groups. Reverse transcriptase polymerase chain reaction analyses showed that Survivin and XIAP mRNA expression in emodin and the combination groups were downregulated significantly compared to the other two groups. Furthermore, the expression of the nuclear transcription factor κB (NF-κB) protein and NF-κB mRNA were downregulated in the emodin and the combination groups. DNA-binding activity of NF-κB was inhibited in emodin and combination groups compared to the other groups. This study suggests that emodin potentiates the antitumor effects of gemcitabine in PANC-1 cell xenografts via promotion of apoptosis and IAP suppression.