Carrimycin Affects Melanoma Proliferation by Regulating Macrophage Polarization

doi:10.13523/j.cb.2203018

China Biotechnology

2022, Vol. 42

Issue (7): 12-23 DOI: 10.13523/j.cb.2203018

Carrimycin Affects Melanoma Proliferation by Regulating Macrophage Polarization

Zi-rong YANG¹,Xuan YANG¹,Ting-ting NI²,Cong PAN³,Shi-sheng TAN^1,^2,^**(

),Zi WANG^1,^2,^**(

)

1. Medicine College, Guizhou University, Guiyang 550025, China
2. Department of Oncology, Guizhou Provincial People’s Hospital, Guiyang 550002, China
3. State Key Laboratory of Biotherapy, Sichuan University, Chengdu 610041, China

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Abstract

To investigate whether carrimycin (CAM) affects the occurrence and development of melanoma by regulating the polarization of macrophages, and the following related cell biology assays were used to examine its function. Methods: The effect of CAM on macrophage polarization was detected by real-time quantitative polymerase chain reaction (Q-RT-PCR) and Western blot. Flow cytometry and Cell Counting Kit-8 were used to detect the effect of CAM on mouse macrophages in vitro and in vivo phagocytosis and proliferation. Cell line-derived xenograft model was constructed via B16-F10 to evaluate the anti-tumor effect of CAM on melanoma. Results: In the mRNA level, CAM could up-regulate the levels of TNF-α and iNOS in M1 and down-regulate the level of Arg-1 in M2. In the protein level, CAM can increase the expression of p-STAT1 and decrease the expression of p-STAT3. In the cell line-derived xenograft model, these data shown that the occurrence and CAM development of melanoma was inhibited after CAM treatment, the tumor inhibition rate was 41.6%, and promoted the increase of the number of M1 macrophages (P<0.05). Conclusion: CAM promotes the increase in the number of M1 macrophages in vivo and inhibits the progression of melanoma, suggesting that CAM may achieve anti-tumor effects by inducing the polarization of macrophages to M1.

Key words： Carrimycin Melanoma Anti-tumor Macrophage Polarization

Received: 08 March 2022 Published: 03 August 2022

ZTFLH:

Q813

Corresponding Authors: Shi-sheng TAN,Zi WANG E-mail: wangzi@gz5055.com;tssh18018@126.com

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	Zi-rong YANG
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Cite this article:

Zi-rong YANG,Xuan YANG,Ting-ting NI,Cong PAN,Shi-sheng TAN,Zi WANG. Carrimycin Affects Melanoma Proliferation by Regulating Macrophage Polarization. China Biotechnology, 2022, 42(7): 12-23.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2203018 OR https://manu60.magtech.com.cn/biotech/Y2022/V42/I7/12

Table 1 Q-RT-PCR primers for TNF-α,iNOS,Arg-1,β-actin

Fig.1 CAM’s effects on phagocytosis and proliferation of macrophages in vitro (a) Flow cytometry to measure the phagocytic activity of peritoneal macrophages (b) Cell Counting kit 8 was used to determine the IC₅₀ of CAM in macrophages (c) Cell Counting kit 8 was used to assess the proliferation of macrophages after 12 hours. CAM treatment, n=3, ^* P<0.05

Fig.2 Quantitative-RT PCR analyses the effect of CAM on RAW246.7 cell polarization (a) The effect of CAM on the polarization of M1 (Group A) and M2 macrophages (Group A) The effect of CAM on the repolarization of (b) M1 macrophages (Group B) and (c) M2 macrophages (Method: Group B). NC: RAW cells without any treatment; M1:RAW246.7 cells plus LPS + INF-γ to induce into M1; M2:RAW246.7 cells plus IL-4 to induce into the M2 type. Group A: CAM+M1/CAM+M2 (RAW246.7 cells were treated with CAM, which was followed by LPS+ INF -γ/IL-4); Group B: M1+CAM/M2+CAM (RAW246.7 cells were treated with LPS + INF-γ/IL-4 to promote differentiation into M1/M2, followed by CAM treatment. Unpaired t-tests were performed, n=3, ^* P<0.05, ^** P<0.01

Fig.3 Quantitative-RT PCR analyses the effect of CAM on primary macrophage polarization (a) Validation of primary macrophages (b) The effect of CAM on the polarization of M1 (Group A) and M2 macrophages (Group A) (c) The effect of CAM on the repolarization of M1 macrophages (Group B) (d) The effect of CAM on the repolarization of M2 macrophages (Method: Group B). NC: Primary macrophages without any treatment; M1:Primary macrophages were induced to the M1 type using LPS + INF-γ; M2:Primary macrophages were induced to the M2 type using IL-4. Group A: CAM+M1/CAM+M2 (primary macrophages cells were treated with CAM, and then LPS+ INF-γ/IL-4). Group B: M1+CAM/M2+CAM (primary macrophages were treated with LPS + INF-γ/IL-4 to promote differentiation into the M1/ M2 type, followed by CAM). Unpaired t-tests were performed, n=3, ^* P<0.05, ^** P<0.01

Fig.4 Western blot was used to determine the effect on macrophage polarization pathway protein after CAM treatment (a) The effects of CAM on STAT1 and p-STAT1 protein levels (b) The effects of CAM on STAT3 and p-STAT3 protein levels. This data was expressed using the mean ± the standard error of the mean, n=3, ^* P<0.05, ^** P<0.01, ^*** P<0.001

Fig.5 The effect of CAM on monocytes and macrophage proliferation in vivo Flow cytometry demonstrating the proliferation of monocytes in (a) peripheral blood and (b) peritoneal fluid (c) The median signal value for M1-macrophages in peritoneal fluid. This data was expressed using the mean ± the standard error of the mean, n=5, ^* P<0.05, ^** P<0.01

Fig.6 Antitumor effects of CAM in a murine melanoma model (a) A photograph of subcutaneous tumors arranged in ascending order according to the group; a scatter plot of tumor weight and a statistical analysis (compared with the NC group) (b) A proliferation curve showing the tumor volume in the CDX model (c) Flow cytometry analysis showing the proportion of macrophages in the tumor tissue (d) The median value of the M1signal in the tumor tissue. This data was expressed using the mean ± the standard error of the mean, n=5, ^* P<0.05, ^** P<0.01, ^*** P<0.001


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