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.

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.

The distinct mechanisms of the antitumor activity of emodin in different types of cancer (Review).

Emodin, a tyrosine kinase inhibitor, is a natural anthraquinone derivative found in the roots and rhizomes of numerous plants. The inhibitory effect of emodin on mammalian cell cycle modulation in specific oncogene-overexpressing cells has formed the basis for using this compound as an anticancer drug. Previous reviews have summarized the antitumor properties of emodin. However, the specific molecular mechanisms of emodin-mediated tumor inhibition have not been completely elucidated over the last 5 years. Recently, there has been great progress in the preclinical study of the anticancer mechanisms of emodin. Our recent study revealed that emodin has therapeutic effects on pancreatic cancer through various antitumor mechanisms. Notably, the therapeutic efficacy of emodin in combination with chemotherapy was found to be higher than the comparable single chemotherapeutic regime, and the combination therapy also exhibited fewer side-effects. Despite these encouraging results, further investigation is warranted as emodin has been shown to modulate one or more key regulators of cancer growth. This review provides an overview of the distinct mechanisms of anticancer action of emodin in different body systems identified over the past 5 years. These new breakthrough findings may have important implications for targeted cancer therapy and for the future clinical use of emodin.

Emodin sensitizes the gemcitabine-resistant cell line Bxpc-3/Gem to gemcitabine via downregulation of NF-κB and its regulated targets.

The aim of this study was to evaluate whether emodin can overcome the chemoresistance of the gemcitabine-resistant cancer cell line (Bxpc-3/Gem) in vitro. The cell line Bxpc-3/Gem was derived from the human pancreatic cancer cell line Bxpc-3. We found that Bxpc-3/Gem cells were characterized by a series of morphological changes with a resistance index of 43.51 comparing with the parental cell line. Emodin reduced Bxpc-3/Gem cell proliferation in a dose-dependent manner. Emodin and gemcitabine combination treatments resulted in decreased cell proliferation and increased apoptosis in Bxpc-3/Gem cells. In addition, combination treatments resulted in downregulation of gene and protein expression of MDR-1 (P-gp), NF-κB, XIAP, survivin, as well as inhibition of NF-κB activity and P-gp function. These observations suggest that emodin may sensitize the pancreatic cancer gemcitabine-resistant cell line Bxpc-3/Gem to gemcitabine therapy via inhibition of survival signaling.

Antitumor activity of emodin against pancreatic cancer depends on its dual role: promotion of apoptosis and suppression of angiogenesis.

BACKGROUND: Emodin has been showed to induce apoptosis of pancreatic cancer cells and inhibit tumor growth in our previous studies. This study was designed to investigate whether emodin could inhibit the angiogenesis of pancreatic cancer tissues and its mechanism. METHODOLOGY/PRINCIPAL FINDING: In accordance with our previous study, emodin inhibited pancreatic cancer cell growth, induced apoptosis, and enhanced the anti-tumor effect of gemcitabine on pancreatic caner cells in vitro and in vivo by inhibiting the activity of NF-κB. Here, for the first time, we demonstrated that emodin inhibited tumor angiogenesis in vitro and in implanted pancreatic cancer tissues, decreased the expression of angiogenesis-associated factors (NF-κB and its regulated factors VEGF, MMP-2, MMP-9, and eNOS), and reduced eNOS phosphorylation, as evidenced by both immunohistochemistry and western blot analysis of implanted tumors. In addition, we found that emodin had no effect on VEGFR expression in vivo. CONCLUSIONS/SIGNIFICANCE: Our results suggested that emodin has potential anti-tumor effect on pancreatic cancer via its dual role in the promotion of apoptosis and suppression of angiogenesis, probably through regulating the expression of NF-κB and NF-κB-regulated angiogenesis-associated factors.

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.

Enhanced antitumor efficacy of gemcitabine by evodiamine on pancreatic cancer via regulating PI3K/Akt pathway.

Evodiamine has therapeutic potential against cancers. This study was designed to investigate whether combination therapy with gemcitabine and evodiamine enhanced antitumor efficacy in pancreatic cancer. In vitro application of the combination therapy triggered significantly higher frequency of pancreatic cancer cells apoptosis, inhibited the activities of PI3K, Akt, PKA, mTOR and PTEN, and decreased the activation of NF-κB and expression of NF-κB-regulated products. In vivo application of the combination therapy induced significant enhancement of tumor cell apoptosis, reductions in tumor volume, and inhibited activation of mTOR and PTEN. In conclusion, evodiamine can augment the therapeutic effect of gemcitabine in pancreatic cancer through direct or indirect negative regulation of the PI3K/Akt pathway.

Emodin reverses gemcitabine resistance in pancreatic cancer cells via the mitochondrial apoptosis pathway in vitro.

Gemcitabine resistance is a common problem of pancreatic cancer chemotherapy, and how to reverse it plays an important role in the treatment of pancreatic cancer. This study investigated the effect of emodin on the gemcitabine-resistant pancreatic cancer cell line SW1990/Gem, and explored the potential mechanism of its action. SW1990/Gem was obtained by culture of the pancreatic cancer cell line SW1990 in vitro by intermittently increasing the concentration of gemcitabine in the culture medium for 10 months, observing the morphology using inverted microscopy. SW1990/Gem cells were pretreated with emodin (10 µM) for different periods followed by treatment with gemcitabine (20 µM) for 48 h; cell proliferation was tested by MTT assay. SW1990/Gem cells were treated by emodin with different concentrations for 48 h, cell apoptosis was detected by flow cytometry (FCM). The expression of gene and protein, such as MDR-1 (P-gp), NF-κB, Bcl-2, Bax, cytochrome-C (cytosol), caspase-9 and -3 were measured by RT-PCR and Western blotting. The function of P-gp in SW1990/Gem cells was checked by FCM. The results showed that the SW1990/Gem cells changed greatly in morphology and the resistance index was 48.63. Emodin promoted cell apoptosis of the gemcitabine-resistant cell line SW1990/Gem in a dose-dependent manner. Emodin enhanced the SW1990/Gem cell sensitivity to gemcitabine in a time-dependent manner. Emodin monotherapy or combination with gemcitabine both decreased the gene and protein expression levels of MDR-1 (P-gp), NF-κB and Bcl-2 and inhibited the function of P-gp, but increased the expression levels of Bax, cytochrome-C (cytosol), caspase-9 and -3, and promoted cell apoptosis. This demonstrated that emodin had a reversing effect on the gemcitabine-resistant cell line SW1990/Gem, possibly via decreasing the function of P-gp and activating the mitochondrial apoptosis pathway in vitro.

Antitumor and apoptosis-promoting properties of emodin, an anthraquinone derivative from Rheum officinale Baill, against pancreatic cancer in mice via inhibition of Akt activation.

Pancreatic adenocarcinoma is one of the most common malignancies worldwide. Gemcitabine is currently the standard first-line chemotherapeutic agent for pancreatic cancer. However, gemcitabine can induce activation of Akt and nuclear factor-κB (NF-κB), which is associated with its chemoresistance. It has been reported that gemcitabine combination therapies result in improved survival outcomes in pancreatic cancer. Therefore, agents that can either enhance the effects of gemcitabine or overcome chemoresistance to the drug are needed for the treatment of pancreatic cancer. Emodin is an active component of Chinese medicinal herbs and can inhibit the activation of Akt and NF-κB. In this study, we investigated whether emodin could enhance the anticancer effect of gemcitabine on pancreatic cancer in vivo. We demonstrated that treatment of gemcitabine combined with emodin efficiently suppressed tumor growth in mice inoculated with pancreatic tumor cells. This treatment paradigm promoted apoptotic cell death and mitochondrial fragmentation. Furthermore, it reduced phosphorylated-Akt (p-Akt) level, NF-κB activation and Bcl-2/Bax ratio, increased caspase-9 and -3 activation, Cytochrome C (CytC) release occurred in combination therapy. Collectively, emodin enhanced the activity of gemcitabine in tumor growth suppression via inhibition of Akt and NF-κB activation, thus promoting the mitochondrial-dependent apoptotic pathway. Therefore, our findings may provide new insights into understanding the pharmacological regulation of emodin on gemcitabine-mediated proapoptosis in pancreatic cancer and may aid in the design of new therapeutic strategies for the intervention of human pancreatic cancers.