| Backgrounds and objectives Pancreatic cancer is one of the highest mortality tumors. The etiology of pancreatic cancer remains unclear, and the factors including genetics, diet and chronic pancreatitis may contribute to the occurrence and development of pancreatic cancer. Due to the changes of living fashion, dietary structure and environmental pollution, the incidence of pancreatic cancer has a tendency to increase gradually. Five-year survival rate of pancreatic cancer is extremely low, because of the strong growth of pancreatic cancer cells, early local transmission and transfer capability, resulting in hindering in the radiotherapy and systemic chemotherapy. The lack of effective early diagnosis way may be the main reason that leading to unexpected pancreatic cancer. The most patients are found in the late phase of cancer. Clinically, surgical operation is regarded as the main way to treat pancreatic cancer. However, in fact, only a small number of patients with pancreatic cancer can receive the treatment of surgical operation. Other patients with pancreatic cancer who are unable to undergo the surgery presently used to be treated with 5-fluorouracil, cisplatin, gemcitabine, or a combination of radiation therapy and chemical therapy.Although these auxiliary treatments can to some extent inhibit the further deterioration in patients with pancreatic cancer, but whether they can extend the life of the patients need yet to be clinical observation for a long time. Thus, it is an urgent to develop new treatment and effective drugs for pancreatic cancer. The Hedgehog signaling is a conservative pathway involved in embryonic development. Abnormal activation of hedgehog signaling is associated with a variety of tumors, especially with the occurrence and development of pancreatic cancer. The activation of Hedgehog pathway can induce cell cycle disorder, promote cell proliferation and inhibit apoptosis. As a result, pancreatic cells become cancerous. Sedum sarmentosum Bunge(SSB) is a commonly used traditional Chinese medicine which is included in “Chinese pharmacopoeia”. SSB belongs to the crassulaceae stonecrop perennial herbaceous plants. As a kind of important medicinal plant resource, SSB extract(SSBE) clinically is mainly used for hot and humid yellow gangrene, urination and carbuncle swollen ulcers. In addition, SSBE has also been shown to have activities on immunosuppression, anti-inflammation and anti-cancer of liver tissue. However, the role of SSBE in the development of pancreatic cancer remains unknown. In the present study, the effects of SSBE on the biological behavior of pancreatic cancer cells, including the proliferation and apoptosis, cell cycle, and migration ability were investigated. In addition, the effects of SSBE on the Hedgehog signaling pathway in pancreatic cancer cells were also evaluated. Furthermore, the flavonoid components of SSBE were analyzed in order to make clear the molecular mechanisms of these ingredients in the development of pancreatic cancer. Our study not only provides new ideas for targeted therapy of pancreatic cancer, but also accumulates experimental basis to the research of mechanism of pancreatic cancer development.Methods 1. Effects of SSBE on the proliferation and apoptosis of pancreatic cancer cells: The morphology of the PANC-1 cells was observed under inverted/phase contrast microscope. The effects of SSBE on the proliferation and vitality of PANC-1 cells were examined by CCK-8 assay. The effects of SSBE on cellular apoptosis were determined by flow cytometry(FCM) assay using an Annexin V-FITC staining. Protein expression of caspase-3, caspase-8, bcl-2, bax, c-myc, and p53 were quantified by Western blot analysis. The m RNA expression of Bcl-2, Bax, c-Myc, TP53, and Survivin were also quantified by q RT-PCR analysis. Immunofluorescent staining was performed to detect the expression of c-Myc and proliferating cell nuclear antigen(PCNA). 2. Effects of SSBE on cell cycle phase distribution of pancreatic cancer cells: Cell cycle of pancreatic cancer was analyzed by flow cytometry assay. Western blot analysis was used to examine the protein expression of p21(Waf1/CIP1), and q RT-PCR was used to examine the m RNA level of CCDN-1(expressed cyclin D1). 3. Effects of SSBE on epithelial-mesenchymal transition(EMT) of pancreatic cancer cells: q RT-PCR analysis and immunofluorescent staining were performed to detect the m RNA and protein expression of E-cadherin and α-smooth muscle actin(α-SMA), respectively, in PANC-1 cells treated with SSBE. 4. Effects of SSBE on tumor growth in animal xenograft models of pancreatic cancer: The left neck of experimental nude mice(BALB/c, n = 24) were injected subcutaneously with 5 × 106 PANC-1 cells and then received daily intragastric administration of SSBE(10 and 100 mg/kg·d, and each for 12) for one month. Model mice(n = 12) received injection of 5 × 106 PANC-1 cells only, and the healthy group(n = 12) received daily gastric tube of solvent. Tumors were monitored daily until they became cumbersome or necrotic. Tumor volumes were measured every other day. The tumor specimens were stained with hematoxylin and eosin(HE). 5. Effects of SSBE on the activity of Hedgehog signaling in PANC-1 cells: q RT-PCR was used to examine the m RNA expression of Ptch1, Smo and Gli1 in SSBE-treated PANC-1 cells. Immunofluorescent staining was performed to detect the effect of SSBE on the protein expression of Ptch1 and Smo in PANC-1 cells. 6. Effects of SSBE on the activity of Hedgehog signaling in animal xenograft models of pancreatic cancer: In animal xenograft models of pancreatic cancer, immunofluorescent staining was performed to detect the protein expression of Ptch1 and Smo. In addition, q RT-PCR analysis was used to examine the m RNA expression of Ptch1, Smo and Gli1. 7. Effect of the Hedgehog signaling on SSBE-mediated anti-pancreatic cancer activity: immunofluorescent staining was performed to detect the protein expression of Ptch1, Smo, p27(Kip1), and α-SMA in PANC-1 cells treated with SSBE with or without sonic hedgehog(Shh). 8. SSBE components were analyzed using a high performance liquid chromatography(HPLC) method: By comparing the area of the corresponding chromatographic peak in HPLC value between different standard and SSBE test samples, the content of each component in SSBE was detected. 9. Effect of quercetin on the proliferation and apoptosis of pancreatic cancer: The morphology of the PANC-1 cells was observed under inverted/phase contrast microscope. The effects of quercetin on the proliferation and vitality of PANC-1 cells were examined by CCK-8 assay. Immunofluorescent staining was performed to detect the expression of Rac1. Protein expression of p53 was quantified by Western blot analysis. 10. Effect of quercetin on the activity of Hedgehog signaling in PANC-1 cells: Immunofluorescent staining was performed to detect the protein expression of Ptch1, Smo and Gli1 in PANC-1 cells treated with quercetin.Results 1. SSBE induced marked apoptosis and necrosis of PANC-1 cells, and increased the number of early and late apoptotic cells and necrotic cells. SSBE treatment increased the m RNA and protein expression of bax, caspase-3 and caspase-8(P < 0.05), and decreased the m RNA and protein expression of bcl-2(P < 0.05), suggesting that the mitochrondial pathway may be involved in the apoptosis of PANC-1 cells with SSBE treatment. In addition, down-regulated expression of Survivin m RNA were observed in SSBE-treated PANC-1 cells(P < 0.05), indicating that the inhibition in expression of anti-apoptotic protein may be one reason for the pro-apoptotic activity of SSBE on pancreatic cancer cells. 2. SSBE inhibited the proliferation of PANC-1 cells in a concentration-dependent manner. In addition, down-regulated m RNA and protein expression of c-Myc(P < 0.05) and up-regulated m RNA and protein expression of p53(P < 0.05) were also observed in PANC-1 cells with SSBE treatment. Moreover, SSBE reduced the expression of PCNA(P < 0.05). These findings suggested that SSBE exerts its inhibitory effect on the proliferation in PANC-1 cells. 3. The number of G0/G1-phase PANC-1 cells in SSBE-treated PANC-1 cells was significantly decreased compared with those in the controls(P < 0.05), and the number of G2/M-phase cells was increased(P < 0.05). Further study showed that SSBE-induced cell cycle arrest of PANC-1 cells was through down-regulating the m RNA expression of cyclin D1(P < 0.05) and up-regulating the protein expression of cell cycle inhibitor p21(P < 0.05). These results showed that SSBE treatment induced cell cycle arrest of PANC-1 cells at the G2/M phase. 4. The m RNA and protein expressions of α-SMA were significantly down-regulated in PANC-1 cells by SSBE treatment compared with the control(P < 0.05). Additionally, the m RNA and protein expressions of E-cadherin were markedly up-regulated in SSBE-treated cells(P < 0.05). These findings indicated that SSBE inhibited the EMT process of PANC-1 cells. 5. The nude mice received the injection subcutaneously with 5 × 106 PANC-1 cells, and one month later, there occurs obvious protrusion. HE staining identified pathological results of pancreatic cancer in tissues of model group. The mass of transplanted tumor in model group was(122.0 ± 14.8) mg. In SSBE-treated models, the tumor growth was slow. The mass of transplantated tumor in SSBE(10 and 100 mg/kg-1·d)-treated models was(29.5 ± 6.3) mg and(25.3 ± 4.5) mg, respectively. Compared with the transplantated tumor group, the differences of mass of transplantation tumor in SSBE-treated models were significant(P < 0.05). Thus, these findings suggested that SSBE exerted its inhibitory effect on tumor growth. 6. SSBE treatment increased the m RNA and protein expression of Ptch1(P < 0.05), and decreased the m RNA and protein expression of Smo(P < 0.05). In addition, SSBE also inhibited the m RNA expression of Gli1(P < 0.05). These findings suggested that the activity of Hedgehog pathway in PANC-1 cells was reduced by SSBE treatment. 7. Compared with the normal group, the m RNA expression of Ptch1 in transplanted tumor model group was significantly decreased(P < 0.05), and the expression of Smo was increased(P < 0.05), indicating that the Hedgehog signaling was activated in the occurrence and development of pancreatic cancer. SSBE administration significantly enhanced the m RNA expression of Ptch1, and reduced the m RNA expression of Smo and Gli1. These findings indicated that the activity of Hedgehog signaling in transplanted tumor was inhibited by SSBE treatment. 8. Recombinant protein Shh abolished in part SSBE-mediated up-regulated expression of Ptch1(P < 0.05) and down-regulated expression of Gli1(P < 0.05) in PCNA cells. In addition, Shh reversed SSBE-mediated up-regulated expression of cell cycle-regulating protein p27(P < 0.05) and down-regulated expression of mesenchymal marker α-SMA(P < 0.05). These results indicated that Shh enhanced the activity of Hedgehog signaling, and resulted in the ablolishment in part of anti-pancreatic cancer of SSBE. 9. There are five flavonoid components in SSBE, including quercetin, isorhamnetin, kaempfeide, luteolin, and isoliquiritigenin, respectively. Among them, the highest content of SSBE was quercetin with 1.272 mg/g, followed by isorhamnetin with 0.697 mg/g, luteolin with 0.556 mg/g, kaempfeide with 0.355 mg/g, and isoliquiritigenin with 0.028 mg/g. 10. Quercetin markedly induced the apoptosis and necrosis of PANC-1 cells. Quercetin inhibited the proliferation of PANC-1 cells in a concentration- and time-dependent manner. Quercetin treatment decreased the protein expression of Rac1(P < 0.05). Western blot analysis showed that quercetin decreased the protein expression of p53(P < 0.05). These results identified the anti-pancreatic cancer effect of quercetin. 11. Quercetin induced the expression of Ptch1(P < 0.05), and inhibited the expression of Smo and Gli1(P < 0.05), indicating that quercetin exerts the inhibitory effect on the activity of Hedgehog signaling pathway, which were consistent with the effect of SSBE.Conclusion 1. SSBE exerts its anti-pancreatic cancer activity, including(i) regulating the expression of cell cycle-related proteins, and thereby results in cell cycle arrest of pancreatic cancer cells;(ii) inhibiting the proliferation of pancreatic cancer cells, and inducing apoptosis;(iii) inhibiting the EMT process of pancreatic cancer cells, and reducing their invasive abilities;(iv) suppressing the growth of the subcutaneous transplantation tumor. 2. Our in vivo and in vitro experiments showed that down-regulation in the activity of Hedgehog signaling is involved in the anti-pancreatic cancer activity of SSBE. 3. There are five flavonoid components in SSBE, including quercetin, isorhamnetin, kaempfeide, luteolin, and isoliquiritigenin, respectively. Among them, the highest content is quercetin. 4. Quercetin can inhibit the proliferation of pancreatic cancer cells and reduce the migration activity, and thereby exerts its anti-pancreatic cancer effect. The molecular mechanism of Quercetin is associated with the reduction of Hedgehog signaling activity, and quercetin may be main active component of SSBE. |
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