Asiatic acid enhances intratumor delivery and the antitumor effect of pegylated liposomal doxorubicin by reducing tumor-stroma collagen

Abstract

Tumor-targeted drug delivery systems (Tt-DDSs) are proposed as a promising strategy for cancer care. However, the dense collagen network in tumors stroma significantly reduces the penetration and efficacy of Tt-DDS. In order to investigate the effect of asiatic acid (AA) on antitumor effect of pegylated liposomal doxorubicin (PLD) by attenuating stroma-collagen, colon cancer xenograft mice (SW620 cell line) were treated by PLD, AA, or combined regimes, respectively; the collagen levels were estimated by Sirius red/fast green dual staining and immunohistochemistry (IHC) staining; the intratumor exposure of doxorubicin was visualized by ex vivo fluorescence imaging and quantified by HPLC/MS analysis. In addition, the impact of AA on collagen synthesis of fibroblast cell (HFL-1) and cytotoxic effect of PLD and doxorubicin to cancer cell (SW620) were studied in vitro. In the presence of AA (4 mg/kg), the intratumor collagen level was restricted in vivo (reduced by 22%, from 4.14% ± 0.30% to 3.24% ± 0.25%, P = 0.051) and in vitro. Subsequently, doxorubicin level was increased by ~30%. The antitumor activity of PLD was significantly improved (57.3% inhibition of tumor growth and 44% reduction in tumor weight) by AA combination. Additionally, no significant improvement in cytotoxic effect of PLD or doxorubicin induced by AA was observed. In conclusion, AA is a promising sensitizer for tumor treatment by enhancing intratumor drug exposure via stromal remodeling.

Keywords: asiatic acid; colon cancer; intratumor drug delivery; pegylated liposomal doxorubicin; stromal collagen.

Fig. 1

Schedule of treatment

Fig. 2

Antitumor effect of combined treatment with PLD and AA in SW620 xenograft nude mice and SW620 cell line. In vivo, the combined regime of PLD and AA displayed an enhanced antitumor effect with smaller tumor volume (a, b), lower tumor weight (c), and comparable bodyweight (d) compared with PLD (4 mg/kg) alone. However, in vitro, no significant improvement was observed in the cytotoxic effect of PLD (e) and DOX (f) by AA

Fig. 3

Doxorubicin accumulation in SW620 xenograft tumor. (a) Ex vivo fluorescence imaging. The intensity of doxorubicin in the HD-AA combined group was significantly increased compared with mono-PLD treatment. (b) Concentration of doxorubicin quantified by HPLC-MS/MS. The intratumor level of doxorubicin was significantly increased by combination with high-dosage AA (4 mg/kg, red diamond) compared with mono-PLD treatment (black square, P = 0.005), whereas low-dosage AA (1 mg/kg, pink star) failed to enhance intratumor exposure of doxorubicin

Fig. 4

Intratumor-stroma alleviation by AA in SW620 xenograft mouse models. (a) H&E staining and IHC staining of tumor sections treated with PBS and AA (4 mg/kg). The fraction of stroma and collagen IV was reduced in the AA-treated group compared with the PLD-treated group. (b) Relative fraction of total collagen proteins in tumor tissue evaluated using the semi-quantitative kit. AA significantly reduced the level of collagen IV. (c) A correlation between the intratumor level of doxorubicin and total collagen was observed (r = 0.663, P = 0.007)

Fig. 5

AA reversed the increased mRNA and protein levels of COL1A1 and COL4A1 in HFL-1 cells induced by SW620-derived conditioned medium. The mRNA levels of COL1A1 and COL4A1 were analyzed using fluorescent quantitative RT-PCR (a). Error bars represent standard deviations (SDs). Type I and IV collagen proteins were detected by Western blotting (b) and visualized by immunofluorescence (c). Scale bar, 25 μm