The suppressive effect of baicalin on hepatocellular carcinoma through mediating tumour associated macrophages repolarization

Abstract

High aggressiveness and resistance of hepatocellular carcinoma (HCC) renders the inefficient of current therapies in completely eradicating the tumor cells. The aggressive phenotype of HCC is partially attributed by the communication between tumor cells with stromal cells in microenvironment. The tumor microenvironment has been revealed as the prognostic implication for HCC progression, and tumor associated macrophages (TAM) in particular is one of the effectors in HCC microenvironment. Aiming at TAM may thus be a promising strategy for HCC management. Scutellaria baicalensis is one of the most commonly employed herbs for tumor control in traditional Chinese medicinal practice and baicalin is its feature component. However, the in vitro and in vivo HCC inhibitory effect of baicalin remains scanty. Meanwhile, the immuno-modulatory effect of baicalin has been recently underscored. Thus, whether baicalin induces HCC regression via its immuno-modulatory effect and the underlying mechanism worth to be further explored. In this study, we aimed to systematically investigate the in vivo anti-tumour effect of baicalin and the role of TAM in baicalin-mediated tumour inhibition. Firstly, we examined the in vivo effect of baicalin using (i) orthotopic HCC implantation and (ii) choline-deficient L-amino acid defined diet and carbon tetrachloride induced liver carcinogenic murine model. We observed reduced HCC orthotopic growth with no observable toxicity to immuno-compromised mice. The effect was accompanied with increased M1 macrophage population and minimal inflammatory monocytes infiltration. Also, deceleration of liver tumor growth was observed in baicalin-intervened 6- and 9-month diet-fed immuno-competent mice. It was followed with increased ratio of intrahepatic M1 to M2 macrophages and cytotoxic 〖CD〗^(8+) cytotoxic T populations. Surprisingly, we did not observe any inhibition of baicalin on HCC cellular functions. The involvement of macrophages in baicalin-mediated HCC suppression was confirmed by macrophage deletion using liposome clodronate. Subsequently, the effect of baicalin on TAMs functions was systematically investigated using the optimized in vitro culture systems. Our findings postulate that high dose of baicalin induced no specific cytotoxicity to macrophages. Whereas it reduced in vitro monocyte-to-macrophage differentiation and C-C chemokine receptor type-2 (CCR2) expression, a crucial element for monocyte recruitment. Besides, baicalin intervention phenotypically and functionally shifted macrophages towards M1-like, away from M2 in dose-dependent manner. Further co-culturing of baicalin-pre-treated macrophages with Hepa1-6 cells declined survival and motility of Hepa1-6. Lastly, we sought to delineate the mechanism of baicalin-mediated TAMs polarization. Baicalin intervention induced autophagy in macrophages. Autophagy deactivation using pharmacological inhibitor balfilomycin A1 and RNA interference against Atg5 abolished baicalin-mediated macrophage repolarization. Baicalin also promoted IKKα-dependent RelB/p52 activation. While autophagy blockade triggered loss of induction of IKKα and RelB. Baicalin-mediated activation of IKKα-RelB-p52 cascade is negatively regulated by TRAF2. Autophagic degradation of TRAF2 by baicalin was confirmed following observation of recruitment of ubiquitinated TRAF2 to autophagosome. In summary, the study underscores the crucial role of TAM reprogramming mediated HCC inhibition by baicalin, which relying on autophagy-regulated RelB/p52 activation. Further clinical investigation on human safety and efficacy of baicalin may offer novel immune-targeted drug candidate for HCC treatment.