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. 2017 Apr;13(4):2330-2336.
doi: 10.3892/ol.2017.5730. Epub 2017 Feb 13.

Inhibition of lung cancer growth by HangAmDan-B is mediated by macrophage activation to M1 subtype

Hye-Rin Park  1 , Eun-Ji Lee  1 , Seong-Cheol Moon  1 , Tae-Wook Chung  1 , Keuk-Jun Kim  2 , Hwa-Seung Yoo  3 , Chong-Kwan Cho  3 , Ki-Tae Ha  1
Affiliations

Inhibition of lung cancer growth by HangAmDan-B is mediated by macrophage activation to M1 subtype

Hye-Rin Park et al. Oncol Lett. 2017 Apr.

Abstract

Re-education of tumor-associated macrophages (TAMs) toward antitumor effectors may be a promising therapeutic strategy for the successful treatment of cancer. HangAmDan-B (HAD-B), a herbal formula, has been used for stimulating immune function and activation of vital energy to cancer patients in traditional Korean Medicine. Previous studies have reported the anti-angiogenic and anti-metastatic effects of HAD-B; however, evidence on the immunomodulatory action of HAD-B was not demonstrated. In the present study, immunocompetent mice were used to demonstrate the suppression of the in vivo growth of allograft Lewis lung carcinoma (LLC) cells, by HAD-B. In addition, HAD-B inhibited the in vitro growth of LLC cells by driving macrophages toward M1 polarization, but not through direct inhibition of tumor cell growth. Furthermore, culture media transfer of HAD-B-treated macrophages induced apoptosis of LLC cells. Results of the present study suggest that the antitumor effect of HAD-B may be explained by stimulating the antitumor function of macrophages. Considering the importance of re-educating TAMs in the regulation of the tumor microenvironment, the present study may confer another option for anti-cancer therapeutic strategy, using herbal medicines such as HAD-B.

Keywords: HangAmDan-B; M1; apoptosis; lung cancer; macrophage.

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Figures

Figure 1.
Figure 1.
HAD-B inhibits tumor growth in in vivo allograft mouse model using LLC cells. LLC cells (5×105 cells/100 µl PBS) were subcutaneously injected into immunocompetent C57BL/6 mice. HAD-B (2 mg/100 µl PBS) or carrier only were orally administrated for 21 days. (A) After 14, 17 and 21 days, the tumor size was measured using calipers and the volume was calculated. The results were shown as the mean ± SD. (B) At the end of experiment, the tumor was excised and weighed. The data were presented as the mean ± SD. (C) Images of representative tumor samples from control and HAD-B groups were shown. (D) The diameters of excised tumors were measured using calipers and the data were presented as the mean ± SD. *P<0.05, **P<0.01 vs. control. LLC, Lewis lung carcinoma; HAD-B, HangAmDan-B; SD, standard deviation; con, control.
Figure 2.
Figure 2.
HAD-B suppresses growth of LLC cells macrophage-dependent manners. (A) The LLC cells were treated with the indicated concentrations of HAD-B for 24 or 48 h. The viabilities of LLC cells were estimated by MTT assay. The results were calculated by percentage of control. (B) RAW 264.7 cells were treated with the indicated concentration of HAD-B for 24 h. The viabilities of RAW 264.7 cells were measured using a MTT assay and calculated as a percentage of the control. (C) RAW 264.7 cells were treated with the indicated concentrations of HAD-B for 24 h, and the culture media were transferred to LLC cells. Following incubation for 24 or 48 h, the viabilities of LLC cells were measured using a MTT assay. The results were calculated as a percentage of the control and shown as the mean ± standard deviation. **P<0.01, ***P<0.001 compared with the control. HAD-B, HangAmDan-B; LLC, Lewis lung carcinoma; SD, standard deviation; RAW-CM, culture media of RAW 264.7 cells; ns, not significant.
Figure 3.
Figure 3.
HAD-B increased the expression of M1 markers of macrophages. (A) RAW 264.7 cells were treated with the indicated concentrations of HAD-B for 24 h. The expression of M1 and M2 markers were estimated by reverse transcription-polymerase chain reaction analysis. The expression of GAPDH was used for internal control. (B) Morphological changes of RAW 264.7 cells treated with the indicated concentrations of HAD-B were observed by inverted optical microscopy (magnification; ×200). The representative images are shown. HAD-B, HangAmDan-B; iNOS, inducible nitric oxide synthase; IL-1β, interleukin-1β; MCP-1, monocyte chemoattractant protein-1; TNF-α, tumor necrosis factor-α; Arg-1, arginase-1; YM1, yamaha 1; CD206, cluster of differentiation 206.
Figure 4.
Figure 4.
HAD-B induces apoptosis of LLC cells through soluble factors secreted by macrophages. RAW 264.7 cells were treated with the indicated concentration of HAD-B for 24 h. The cultured media were transferred to LLC cells and incubated for 24 h. (A) Apoptosis of LLC cells was detected by fluorescence-activated cell sorting analysis using Annexin V-FITC and propidium iodide double-staining. (B) The activations of caspase-3 and PARP were measured by western blot analysis. HAD-B, HangAmDan-B; Lewis LLC, lung carcinoma; PARP, poly-ADP-ribose polymerase; FITC, fluorescein isothiocyanate.
Figure 5.
Figure 5.
Schematic representation of the mechanism underlying antitumor action of HAD-B. Although HAD-B itself did not show any inhibitory effect on tumor cell growth, tumor cell growth was reduced by HAD-B treatment in the in vivo and in vitro experiments. The culture media from HAD-B-treated macrophages can suppress tumor growth. In addition, HAD-B polarizes macrophages to a M1 phenotype. Thus, the soluble factors secreted from HAD-B-treated macrophages can induce apoptosis of tumor cells. PARP, poly-ADP-ribose polymerase.
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