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Luteoloside suppresses proliferation and metastasis of hepatocellular carcinoma cells by inhibition of NLRP3 inflammasome.

Fan SH, Wang YY, Lu J, Zheng YL, Wu DM, Li MQ, Hu B, Zhang ZF, Cheng W, Shan Q - PLoS ONE (2014)

Bottom Line: Further studies showed that luteoloside could significantly reduce the intracellular reactive oxygen species (ROS) accumulation.The decreased levels of ROS induced by luteoloside was accompanied by decrease in expression of NLRP3 inflammasome resulting in decrease in proteolytic cleavage of caspase-1.Inactivation of caspase-1 by luteoloside resulted in inhibition of IL-1β.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province, School of Life Science, Jiangsu Normal University, Xuzhou, Jiangsu, China.

ABSTRACT
The inflammasome is a multi-protein complex which when activated regulates caspase-1 activation and IL-1β secretion. Inflammasome activation is mediated by NLR proteins that respond to stimuli. Among NLRs, NLRP3 senses the widest array of stimuli. NLRP3 inflammasome plays an important role in the development of many cancer types. However, Whether NLRP3 inflammasome plays an important role in the process of hepatocellular carcinoma (HCC) is still unknown. Here, the anticancer effect of luteoloside, a naturally occurring flavonoid isolated from the medicinal plant Gentiana macrophylla, against HCC cells and the underlying mechanisms were investigated. Luteoloside significantly inhibited the proliferation of HCC cells in vitro and in vivo. Live-cell imaging and transwell assays showed that the migration and invasive capacities of HCC cells, which were treated with luteoloside, were significantly inhibited compared with the control cells. The inhibitory effect of luteoloside on metastasis was also observed in vivo in male BALB/c-nu/nu mouse lung metastasis model. Further studies showed that luteoloside could significantly reduce the intracellular reactive oxygen species (ROS) accumulation. The decreased levels of ROS induced by luteoloside was accompanied by decrease in expression of NLRP3 inflammasome resulting in decrease in proteolytic cleavage of caspase-1. Inactivation of caspase-1 by luteoloside resulted in inhibition of IL-1β. Thus, luteoloside exerts its inhibitory effect on proliferation, invasion and metastasis of HCC cells through inhibition of NLRP3 inflammasome. Our results indicate that luteoloside can be a potential therapeutic agent not only as an adjuvant therapy for HCC, but also, in the control and prevention of metastatic HCC.

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Luteoloside inhibits tumorigenic and spontaneous lung metastatic capabilities of SMMC-7721 cells.(A) Subcutaneous injection of SMMC-7721 cells plus luteoloside treatment in nude mice inhibited tumor growth. (B) Tail vein injection of SMMC-7721 cells plus luteoloside treatment in nude mice inhibited the metastasis of SMMC-7721 cells. (a) 2×106 SMMC-7721 cells were subcutaneously injected into the right upper flank of each mouse. When tumors were observable, the animals were equally divided into two groups (ten per group). The first group received only 0.2 ml of vehicle material by gavage daily and served as a control group. The second group of animals received luteoloside (2 mg/kg body weight) in vehicle, respectively, for 4 weeks. At the termination of the experiment, the mice were sacrificed, and the tumors were weighed immediately after dissection. The yellow arrow shows the tumor. (b) The photo of tumors isolated from killed nude mice of the indicated groups. (c-d) The volume and weight of the tumors. (e) Number of metastatic nodules on the surface of the lungs of mice injected with SMMC-7721 (n = 10 mice in per group) are presented as the means and SEM. (f-g) Representative pictures of lungs with or without metastatic nodules are shown (H&E staining). *, P<0.05; **, P<0.01; ***, P<0.001, versus non-luteoloside-treated control group. Scale bar: 30 µm.
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pone-0089961-g005: Luteoloside inhibits tumorigenic and spontaneous lung metastatic capabilities of SMMC-7721 cells.(A) Subcutaneous injection of SMMC-7721 cells plus luteoloside treatment in nude mice inhibited tumor growth. (B) Tail vein injection of SMMC-7721 cells plus luteoloside treatment in nude mice inhibited the metastasis of SMMC-7721 cells. (a) 2×106 SMMC-7721 cells were subcutaneously injected into the right upper flank of each mouse. When tumors were observable, the animals were equally divided into two groups (ten per group). The first group received only 0.2 ml of vehicle material by gavage daily and served as a control group. The second group of animals received luteoloside (2 mg/kg body weight) in vehicle, respectively, for 4 weeks. At the termination of the experiment, the mice were sacrificed, and the tumors were weighed immediately after dissection. The yellow arrow shows the tumor. (b) The photo of tumors isolated from killed nude mice of the indicated groups. (c-d) The volume and weight of the tumors. (e) Number of metastatic nodules on the surface of the lungs of mice injected with SMMC-7721 (n = 10 mice in per group) are presented as the means and SEM. (f-g) Representative pictures of lungs with or without metastatic nodules are shown (H&E staining). *, P<0.05; **, P<0.01; ***, P<0.001, versus non-luteoloside-treated control group. Scale bar: 30 µm.

Mentions: The results obtained from in vitro studies showed that treatment of HCC cells with luteoloside inhibits the proliferation, migration and invasion capacity of these cells. To determine the in vivo effects of luteoloside, we performed in vivo proliferation and metastasis study. The average size and weight of xenografts in the luteoloside-treated group were dramatically smaller and lighter than those of the control group (P = 0.0026 and P = 0.0417, respectively). (Fig. 5c, 5d). Therefore, the luteoloside treatment significantly inhibited the growth of the xenograft, with inhibition rates (versus the control volume and weight of the tumors) of 44.1 and 53.1%, respectively. Furthermore, we injected SMMC-7721 cells into the lateral tail veins of nude mice (n = 10) and evaluated the metastatic growth of cells in the lung. After 8 weeks, the luteoloside-treated mice displayed a statistically significantly lower number of lung metastases than the control group mice (P = 0.0003), indicative of extravasation and tumor growth in the lung (Fig. 5e). When lungs underwent hematoxylin and eosin staining, lung metastases were observed in all ten mice intravenously injected SMCC-7721 cells only, whereas no obvious lung metastases were observed in the mice intravenously injected SMMC-7721 cells with luteoloside treated (Fig. 5f, 5g). It is worth noting that no difference in mouse weight was observed between the treatment group and the control group, suggesting that luteoloside has no adverse effects on mouse growth.


Luteoloside suppresses proliferation and metastasis of hepatocellular carcinoma cells by inhibition of NLRP3 inflammasome.

Fan SH, Wang YY, Lu J, Zheng YL, Wu DM, Li MQ, Hu B, Zhang ZF, Cheng W, Shan Q - PLoS ONE (2014)

Luteoloside inhibits tumorigenic and spontaneous lung metastatic capabilities of SMMC-7721 cells.(A) Subcutaneous injection of SMMC-7721 cells plus luteoloside treatment in nude mice inhibited tumor growth. (B) Tail vein injection of SMMC-7721 cells plus luteoloside treatment in nude mice inhibited the metastasis of SMMC-7721 cells. (a) 2×106 SMMC-7721 cells were subcutaneously injected into the right upper flank of each mouse. When tumors were observable, the animals were equally divided into two groups (ten per group). The first group received only 0.2 ml of vehicle material by gavage daily and served as a control group. The second group of animals received luteoloside (2 mg/kg body weight) in vehicle, respectively, for 4 weeks. At the termination of the experiment, the mice were sacrificed, and the tumors were weighed immediately after dissection. The yellow arrow shows the tumor. (b) The photo of tumors isolated from killed nude mice of the indicated groups. (c-d) The volume and weight of the tumors. (e) Number of metastatic nodules on the surface of the lungs of mice injected with SMMC-7721 (n = 10 mice in per group) are presented as the means and SEM. (f-g) Representative pictures of lungs with or without metastatic nodules are shown (H&E staining). *, P<0.05; **, P<0.01; ***, P<0.001, versus non-luteoloside-treated control group. Scale bar: 30 µm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3935965&req=5

pone-0089961-g005: Luteoloside inhibits tumorigenic and spontaneous lung metastatic capabilities of SMMC-7721 cells.(A) Subcutaneous injection of SMMC-7721 cells plus luteoloside treatment in nude mice inhibited tumor growth. (B) Tail vein injection of SMMC-7721 cells plus luteoloside treatment in nude mice inhibited the metastasis of SMMC-7721 cells. (a) 2×106 SMMC-7721 cells were subcutaneously injected into the right upper flank of each mouse. When tumors were observable, the animals were equally divided into two groups (ten per group). The first group received only 0.2 ml of vehicle material by gavage daily and served as a control group. The second group of animals received luteoloside (2 mg/kg body weight) in vehicle, respectively, for 4 weeks. At the termination of the experiment, the mice were sacrificed, and the tumors were weighed immediately after dissection. The yellow arrow shows the tumor. (b) The photo of tumors isolated from killed nude mice of the indicated groups. (c-d) The volume and weight of the tumors. (e) Number of metastatic nodules on the surface of the lungs of mice injected with SMMC-7721 (n = 10 mice in per group) are presented as the means and SEM. (f-g) Representative pictures of lungs with or without metastatic nodules are shown (H&E staining). *, P<0.05; **, P<0.01; ***, P<0.001, versus non-luteoloside-treated control group. Scale bar: 30 µm.
Mentions: The results obtained from in vitro studies showed that treatment of HCC cells with luteoloside inhibits the proliferation, migration and invasion capacity of these cells. To determine the in vivo effects of luteoloside, we performed in vivo proliferation and metastasis study. The average size and weight of xenografts in the luteoloside-treated group were dramatically smaller and lighter than those of the control group (P = 0.0026 and P = 0.0417, respectively). (Fig. 5c, 5d). Therefore, the luteoloside treatment significantly inhibited the growth of the xenograft, with inhibition rates (versus the control volume and weight of the tumors) of 44.1 and 53.1%, respectively. Furthermore, we injected SMMC-7721 cells into the lateral tail veins of nude mice (n = 10) and evaluated the metastatic growth of cells in the lung. After 8 weeks, the luteoloside-treated mice displayed a statistically significantly lower number of lung metastases than the control group mice (P = 0.0003), indicative of extravasation and tumor growth in the lung (Fig. 5e). When lungs underwent hematoxylin and eosin staining, lung metastases were observed in all ten mice intravenously injected SMCC-7721 cells only, whereas no obvious lung metastases were observed in the mice intravenously injected SMMC-7721 cells with luteoloside treated (Fig. 5f, 5g). It is worth noting that no difference in mouse weight was observed between the treatment group and the control group, suggesting that luteoloside has no adverse effects on mouse growth.

Bottom Line: Further studies showed that luteoloside could significantly reduce the intracellular reactive oxygen species (ROS) accumulation.The decreased levels of ROS induced by luteoloside was accompanied by decrease in expression of NLRP3 inflammasome resulting in decrease in proteolytic cleavage of caspase-1.Inactivation of caspase-1 by luteoloside resulted in inhibition of IL-1β.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory for Biotechnology on Medicinal Plants of Jiangsu Province, School of Life Science, Jiangsu Normal University, Xuzhou, Jiangsu, China.

ABSTRACT
The inflammasome is a multi-protein complex which when activated regulates caspase-1 activation and IL-1β secretion. Inflammasome activation is mediated by NLR proteins that respond to stimuli. Among NLRs, NLRP3 senses the widest array of stimuli. NLRP3 inflammasome plays an important role in the development of many cancer types. However, Whether NLRP3 inflammasome plays an important role in the process of hepatocellular carcinoma (HCC) is still unknown. Here, the anticancer effect of luteoloside, a naturally occurring flavonoid isolated from the medicinal plant Gentiana macrophylla, against HCC cells and the underlying mechanisms were investigated. Luteoloside significantly inhibited the proliferation of HCC cells in vitro and in vivo. Live-cell imaging and transwell assays showed that the migration and invasive capacities of HCC cells, which were treated with luteoloside, were significantly inhibited compared with the control cells. The inhibitory effect of luteoloside on metastasis was also observed in vivo in male BALB/c-nu/nu mouse lung metastasis model. Further studies showed that luteoloside could significantly reduce the intracellular reactive oxygen species (ROS) accumulation. The decreased levels of ROS induced by luteoloside was accompanied by decrease in expression of NLRP3 inflammasome resulting in decrease in proteolytic cleavage of caspase-1. Inactivation of caspase-1 by luteoloside resulted in inhibition of IL-1β. Thus, luteoloside exerts its inhibitory effect on proliferation, invasion and metastasis of HCC cells through inhibition of NLRP3 inflammasome. Our results indicate that luteoloside can be a potential therapeutic agent not only as an adjuvant therapy for HCC, but also, in the control and prevention of metastatic HCC.

Show MeSH
Related in: MedlinePlus