Limits...
Rapamycin Promotes Mouse 4T1 Tumor Metastasis that Can Be Reversed by a Dendritic Cell-Based Vaccine.

Lin TJ, Liang WM, Hsiao PW, M S P, Wei WC, Lin HT, Yin SY, Yang NS - PLoS ONE (2015)

Bottom Line: Suppression of tumor metastasis is a key strategy for successful cancer interventions.However, rapamycin also exhibits immunosuppressant effects and is hence used clinically as an organ transplantation drug.We hypothesized that the immunosuppressive activities of rapamycin might also negatively mediate host immunity, resulting in promotion of tumor metastasis.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Injury Prevention and Control, Taipei Medical University, Taipei, Taiwan, ROC; Department of Neurosurgery, Taipei Medical University-Wan Fang Hospital, Taipei, Taiwan, ROC; Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan, ROC; Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan, ROC; Taiwan International Graduate Program (TIGP), Molecular and Biological Agricultural Sciences Program, Academia Sinica, Taipei, Taiwan, ROC.

ABSTRACT
Suppression of tumor metastasis is a key strategy for successful cancer interventions. Previous studies indicated that rapamycin (sirolimus) may promote tumor regression activity or enhance immune response against tumor targets. However, rapamycin also exhibits immunosuppressant effects and is hence used clinically as an organ transplantation drug. We hypothesized that the immunosuppressive activities of rapamycin might also negatively mediate host immunity, resulting in promotion of tumor metastasis. In this study, the effects of rapamycin and phytochemical shikonin were investigated in vitro and in vivo in a 4T1 mouse mammary tumor model through quantitative assessment of immunogenic cell death (ICD), autophagy, tumor growth and metastasis. Tumor-bearing mice were immunized with test vaccines to monitor their effect on tumor metastasis. We found that intraperitoneal (ip) administration of rapamycin after a tumor-resection surgery drastically increased the metastatic activity of 4T1 tumors. Possible correlation of this finding to human cancers was suggested by epidemiological analysis of data from Taiwan's National Health Insurance Research Database (NHIRD). Since our previous studies showed that modified tumor cell lysate (TCL)-pulsed, dendritic cell (DC)-based cancer vaccines can effectively suppress metastasis in mouse tumor models, we assessed whether such vaccines may help offset this rapamycin-promoted metastasis. We observed that shikonin efficiently induced ICD of 4T1 cells in culture, and DC vaccines pulsed with shikonin-treated TCL (SK-TCL-DC) significantly suppressed rapamycin-enhanced metastasis and Treg cell expansion in test mice. In conclusion, rapamycin treatment in mice (and perhaps in humans) promotes metastasis and the effect may be offset by treatment with a DC-based cancer vaccine.

No MeSH data available.


Related in: MedlinePlus

In vitro effect of shikonin and rapamycin on expression of immunogenic cell death and autophagosome markers in mouse 4T1 mammary carcinoma cells.4T1 cells were treated with test compounds doxorubicin, shikonin and rapamycin at the indicated concentrations for (A), 24 hours or (B), 48 hours. Tumor cell lysate samples were then prepared with a standardized protocol, as described in Materials and Methods, and subjected to western blot analysis. The results are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4591294&req=5

pone.0138335.g001: In vitro effect of shikonin and rapamycin on expression of immunogenic cell death and autophagosome markers in mouse 4T1 mammary carcinoma cells.4T1 cells were treated with test compounds doxorubicin, shikonin and rapamycin at the indicated concentrations for (A), 24 hours or (B), 48 hours. Tumor cell lysate samples were then prepared with a standardized protocol, as described in Materials and Methods, and subjected to western blot analysis. The results are representative of three independent experiments.

Mentions: Damage- or danger-associated molecular pattern molecules (DAMPs) can initiate and perpetuate immune and inflammatory response and are molecular markers of ICD. The processing of cytosolic LC3β-I to LC3β-II is a marker of autophagosome formation. For this study, cellular debris or organelle samples derived from ICD and autophagy of 4T1 mouse mammary carcinoma cells were tested for use as anti-metastasis vaccines (see below). Toward adjuvant application, we also evaluated whether the phytochemical shikonin or rapamycin could incite ICD and/or autophagic activities in 4T1 cells. Since rapamycin has been previously reported [21] to inhibit the proliferation of breast and other tumor cell lines, we were also interested in a possible direct in vitro cytotoxic effect of rapamycin on 4T1 cells. Shikonin, used as a positive control as defined by our previous study,[11] induced a high level of expression of ICD markers in 4T1 cells at 48 h post treatment (Fig 1B). However, it was effective only at the highest concentration (5 μM) when tested 24 h post treatment (Fig 1A). In contrast, little or no rapamycin effects on ICD activity were observed at 24 h post treatment. Besides, rapamycin did not induce a consistant ICD activity in a dose dependent manner, at 48 h post treatment, (Fig 1A and 1B). At 5 μM, shikonin can effectively induce LC3β-II expression (autophagosome assembly/maturation) at 24 h post treatment, whereas little or no effect of rapamycin (even at 30μM) on LC3β-II was detected (Fig 1A). Even at 48 h post-treatment with rapamycin, the expression of LC3β-II activity was only increased at high dosage (5–30 μM), as revealed by western blot assay (Fig 1B). When 4T1 cells were treated with shikonin (5 μM) for 48 h, >80% of cells were killed by the cytotoxicity of shikonin (S1 Fig). In Fig 1B, the decrease of ICD mediators suggests that the specific time period that can confer ICD activity has apparently “passed” already in the shikonin-treated 4T1 tumor cells. In contrast, high level expression of LC3β-II isoforms was still observed in 4T1 cells at 48 h post treatment with shikonin, indicating a high level of autophagosome has accumulated in the dead 4T1 cells. By taking advantage of these results, for the subsequent experiments in our study we hence induced ICD and autophagy activity in tumor cells with shikonin treatment at 5 μM for 24 h and 48 h, respectively. These results support previous reports of the anti-tumor activities against mammary carcinoma.[22, 23] In view of these results, next we conducted an in vivo experiment on metastasis after resection of primary 4T1 tumors in test mice.


Rapamycin Promotes Mouse 4T1 Tumor Metastasis that Can Be Reversed by a Dendritic Cell-Based Vaccine.

Lin TJ, Liang WM, Hsiao PW, M S P, Wei WC, Lin HT, Yin SY, Yang NS - PLoS ONE (2015)

In vitro effect of shikonin and rapamycin on expression of immunogenic cell death and autophagosome markers in mouse 4T1 mammary carcinoma cells.4T1 cells were treated with test compounds doxorubicin, shikonin and rapamycin at the indicated concentrations for (A), 24 hours or (B), 48 hours. Tumor cell lysate samples were then prepared with a standardized protocol, as described in Materials and Methods, and subjected to western blot analysis. The results are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591294&req=5

pone.0138335.g001: In vitro effect of shikonin and rapamycin on expression of immunogenic cell death and autophagosome markers in mouse 4T1 mammary carcinoma cells.4T1 cells were treated with test compounds doxorubicin, shikonin and rapamycin at the indicated concentrations for (A), 24 hours or (B), 48 hours. Tumor cell lysate samples were then prepared with a standardized protocol, as described in Materials and Methods, and subjected to western blot analysis. The results are representative of three independent experiments.
Mentions: Damage- or danger-associated molecular pattern molecules (DAMPs) can initiate and perpetuate immune and inflammatory response and are molecular markers of ICD. The processing of cytosolic LC3β-I to LC3β-II is a marker of autophagosome formation. For this study, cellular debris or organelle samples derived from ICD and autophagy of 4T1 mouse mammary carcinoma cells were tested for use as anti-metastasis vaccines (see below). Toward adjuvant application, we also evaluated whether the phytochemical shikonin or rapamycin could incite ICD and/or autophagic activities in 4T1 cells. Since rapamycin has been previously reported [21] to inhibit the proliferation of breast and other tumor cell lines, we were also interested in a possible direct in vitro cytotoxic effect of rapamycin on 4T1 cells. Shikonin, used as a positive control as defined by our previous study,[11] induced a high level of expression of ICD markers in 4T1 cells at 48 h post treatment (Fig 1B). However, it was effective only at the highest concentration (5 μM) when tested 24 h post treatment (Fig 1A). In contrast, little or no rapamycin effects on ICD activity were observed at 24 h post treatment. Besides, rapamycin did not induce a consistant ICD activity in a dose dependent manner, at 48 h post treatment, (Fig 1A and 1B). At 5 μM, shikonin can effectively induce LC3β-II expression (autophagosome assembly/maturation) at 24 h post treatment, whereas little or no effect of rapamycin (even at 30μM) on LC3β-II was detected (Fig 1A). Even at 48 h post-treatment with rapamycin, the expression of LC3β-II activity was only increased at high dosage (5–30 μM), as revealed by western blot assay (Fig 1B). When 4T1 cells were treated with shikonin (5 μM) for 48 h, >80% of cells were killed by the cytotoxicity of shikonin (S1 Fig). In Fig 1B, the decrease of ICD mediators suggests that the specific time period that can confer ICD activity has apparently “passed” already in the shikonin-treated 4T1 tumor cells. In contrast, high level expression of LC3β-II isoforms was still observed in 4T1 cells at 48 h post treatment with shikonin, indicating a high level of autophagosome has accumulated in the dead 4T1 cells. By taking advantage of these results, for the subsequent experiments in our study we hence induced ICD and autophagy activity in tumor cells with shikonin treatment at 5 μM for 24 h and 48 h, respectively. These results support previous reports of the anti-tumor activities against mammary carcinoma.[22, 23] In view of these results, next we conducted an in vivo experiment on metastasis after resection of primary 4T1 tumors in test mice.

Bottom Line: Suppression of tumor metastasis is a key strategy for successful cancer interventions.However, rapamycin also exhibits immunosuppressant effects and is hence used clinically as an organ transplantation drug.We hypothesized that the immunosuppressive activities of rapamycin might also negatively mediate host immunity, resulting in promotion of tumor metastasis.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Injury Prevention and Control, Taipei Medical University, Taipei, Taiwan, ROC; Department of Neurosurgery, Taipei Medical University-Wan Fang Hospital, Taipei, Taiwan, ROC; Agricultural Biotechnology Research Center, Academia Sinica, Taipei, Taiwan, ROC; Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan, ROC; Taiwan International Graduate Program (TIGP), Molecular and Biological Agricultural Sciences Program, Academia Sinica, Taipei, Taiwan, ROC.

ABSTRACT
Suppression of tumor metastasis is a key strategy for successful cancer interventions. Previous studies indicated that rapamycin (sirolimus) may promote tumor regression activity or enhance immune response against tumor targets. However, rapamycin also exhibits immunosuppressant effects and is hence used clinically as an organ transplantation drug. We hypothesized that the immunosuppressive activities of rapamycin might also negatively mediate host immunity, resulting in promotion of tumor metastasis. In this study, the effects of rapamycin and phytochemical shikonin were investigated in vitro and in vivo in a 4T1 mouse mammary tumor model through quantitative assessment of immunogenic cell death (ICD), autophagy, tumor growth and metastasis. Tumor-bearing mice were immunized with test vaccines to monitor their effect on tumor metastasis. We found that intraperitoneal (ip) administration of rapamycin after a tumor-resection surgery drastically increased the metastatic activity of 4T1 tumors. Possible correlation of this finding to human cancers was suggested by epidemiological analysis of data from Taiwan's National Health Insurance Research Database (NHIRD). Since our previous studies showed that modified tumor cell lysate (TCL)-pulsed, dendritic cell (DC)-based cancer vaccines can effectively suppress metastasis in mouse tumor models, we assessed whether such vaccines may help offset this rapamycin-promoted metastasis. We observed that shikonin efficiently induced ICD of 4T1 cells in culture, and DC vaccines pulsed with shikonin-treated TCL (SK-TCL-DC) significantly suppressed rapamycin-enhanced metastasis and Treg cell expansion in test mice. In conclusion, rapamycin treatment in mice (and perhaps in humans) promotes metastasis and the effect may be offset by treatment with a DC-based cancer vaccine.

No MeSH data available.


Related in: MedlinePlus