Leinal polypeptide promotes NK cells to suppress PCa survival in vitro

Abstract

As an important component in the innate immune system, natural killer (NK) cells have been demonstrated to be clinically associated with prostate cancer (PCa) progression and castration resistance. Therefore, the development of novel agents that may enhance the cytotoxicity of NK cells possesses promising therapeutic applications. In the present study, leinal polypeptide (LP) solution was supplemented into a co-culture system of NK and PCa cells, as it was previously demonstrated that LP are able to activate NK cells, which kill PCa cells based on an MTT cell viability assay. Mechanistic dissection demonstrated that LP enhanced androgen receptor degradation, which resulted in an upregulation of MHC class I polypeptide-related sequence A (MICA) and MICB. In turn, the induced expression of MICA and MICB was able to further trigger NK cell activation, forming a positive loop between NK cells and PCa cells in the presence of LP solution.

Keywords: leinal polypeptide; natural killer cells; prostate cancer.

Figure 1.

LP enhances the cytotoxicity of NK cells to kill PCa cells. (A) Cartoon showing the co-culture system of NK-92MI cells and PCa cells with LP solution. (B) LP enhances the cytotoxic activity of NK-92MI cell against CWR22Rv1 cells and (C) C4-2 cells. PCa cells (targets:T) were seeded into 24-well plate and incubated with NK-92MI cells (effectors:E) at 20:1 ratio (T:E=20:1) with various concentration of LP solution. Cell viability was measured by MTT assay. (D) Cytokine production was stimulated by LP solution. After co-culture, NK-92MI cells were collected for TNFα and IFNr detection using qPCR. RNAs were normalized to GAPDH, *P<0.05, **P<0.01. LP, lienal polypeptide; NK, natural killer; PCa, prostate cancer.

Figure 2.

AR renders LP-mediated NK cytotoxicity to PCa cells. (A and B) AR expression levels were reduced in (A) CWR22Rv1 and (B) C4-2 by NK-92MI cells upon LP stimulation. GAPDH was used as loading control. (C and D) Overexpression of AR in (C) CWR22Rv1 and (D) C4-2 cells could partially attenuate LP-mediated NK-92MI activation. PCa cells were seeded into 24-well plate and incubated with NK-92MI cells at 20:1 ratio with 20 µl/ml LP solution. Cell viability was determined by MTT assay. AR, androgen receptor; LP, lienal polypeptide; NK, natural killer; PCa, prostate cancer.

Figure 3.

MICA and MICB are directly regulated by AR. (A and B) Knockdown of AR enhances the expression levels of MICA and MICB in both (A) CWR22Rv1 and (B) C4-2 cells. RNAs were normalized to GAPDH. (C) Top, AR binding site in the proximal promoters of MICA and MICB. Bottom, ChIP assay showed that AR directly binds to directly binds to the promoters of MICA and MICB. (D) Luciferase based promoter activity assay revealed that AR could suppress the promoter's activities of both MICA and MICB, *P<0.05. MICA, MHC class I polypeptide-related sequence A; AR, androgen receptor.

Figure 4.

MICA and MICB are involved in LP-mediated NK activation. (A) NK-92MI cells could further enhance the expression levels of MICA and MICB in PCa cells when LP solution was supplied into co-culture medium. RNAs were normalized to GAPDH. (B and C) Knockdown of MICA or MICB in (B) C4-2 cells blocked LP-mediated killing ability of (C) NK-92MI cells. (D) Schematic depiction the interaction between NK and PCa cells in the presence of LP solution, *P<0.05, **P<0.01, ***P<0.001. MICA, MHC class I polypeptide-related sequence A; LP, lienal polypeptide; NK, natural killer; PCa, prostate cancer.