Honokiol inhibits in vitro and in vivo growth of oral squamous cell carcinoma through induction of apoptosis, cell cycle arrest and autophagy

Abstract

Honokiol, an active natural product derived from Magnolia officinalis, exerted anticancer effects through a variety of mechanisms on multiple types of cancers. In this study, the molecular mechanisms of honokiol in suppressing the human oral squamous cell carcinoma (OSCC) cells were evaluated. Treatment of two OSCC cell lines with honokiol resulted in reducing the cell proliferation and arresting the cell cycle at G1 stage which was correlated with the down-regulation of Cdk2 and Cdk4 and the up-regulation of cell cycle suppressors, p21 and p27. In addition, the caspase-dependent programmed cell death was substantially detected, and the autophagy was induced as the autophagosome formation and autophagic flux proceeded. Modulation of autophagy by autophagic inducer, rapamycin or inhibitors, 3-MA or bafilomycin, potentiated the honokiol-mediated anti-OSCC effects where honokiol exerted multiple actions in suppression of MAPK pathway and regulation of Akt/mTOR or AMPK pathways. As compared to clinical therapeutic agent, 5-FU, honokiol exhibited more potent activity against OSCC cells and synergistically enhanced the cytotoxic effect of 5-FU. Furthermore, orally administrated honokiol exerted effective antitumour activity in vivo in OSCC-xenografted mice. Thus, this study revealed that honokiol could be a promising candidate in preventing human OSCCs.

Keywords: Honokiol; apoptosis; autophagy; cell cycle arrest; human oral squamous cell carcinoma.

Figure 1

Honokiol inhibited the growth of human OSCC cell lines. Cells were incubated without or with various concentrations of honokiol for 24, 48 and 72 hr, and the cellular viability of (A) OC2 and (B) OCSL cells was analysed by the CCK‐8 assay. All of the data were expressed as mean ± S.D. of three independent experiments.

Figure 2

Honokiol arrested the progress of cell cycle at G0/G1 phase in the human OSCC cell lines. (A) OC2 and (C) OCSL cells were incubated with various concentrations of honokiol for 24 and 48 hr, and the cell cycle stages were determined by flow cytometric analysis. The expression of cyclin D1, Cdk4, p27, cyclin E, Cdk2 and p21 in the (B) OC2 and (D) OCSL cells was analysed by Western blotting, and GAPDH was used as an internal control.

Figure 3

Honokiol‐induced apoptosis in human OSCC cells. (A) OC2 and (B) OCSL cells incubated with honokiol for 24 and 48 hr were harvested and stained with PI/annexin V for flow cytometry. The percentage of apoptotic cells were calculated and plotted. The data present as the mean ± S.D. of three independent experiments. **P < 0.01.

Figure 4

Detection of honokiol‐mediated apoptosis in human OSCC cells. OC2 and OCSL cells treated with various concentrations of honokiol for 24, 48 and 72 hr, and the expression of caspase‐9, caspase‐8, Bid and cleavage forms of caspase‐3 was analysed by Western blotting. GAPDH was used as an internal control.

Figure 5

Autophagy induced by honokiol in human OSCC cells. To clarify that honokiol induced autophagosome formation, an inducer (rapamycin) of autophagy was tested. The expression levels of LC3‐II were determined for various concentrations of rapamycin and honokiol treatment in (A, B) OC2 and (D, E) OCSL cells. (C) OC2 and (F) OCSL cells were treated with honokiol (0 and 40 μM), and then, the cell lysates were collected at 12, 24, 48 and 72 hr for Western blotting of p62 proteins. GAPDH was used as an internal control.

Figure 6

Autophagy was triggered after honokiol treatment in human OSCC cells. (A) OC2 and (B) OCSL cells were treated with DMSO, rapamycin and honokiol for 12 and 18 hr. Cells were stained with anti‐rabbit LC3, and the autophagosome was determined. (C) High‐content image analysis of LC3 puncta in (A) and (B) was determined, respectively. The fluorescence intensity of LC3 was analysed by BD Attovision software. The data present as the mean ± S.D. of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus DMSO.

Figure 7

Signalling pathways modulation by honokiol. After treatment with honokiol, cells were harvested and analysed by Western blotting with antibodies against (A) p38, ERK and JNK, and (B) AMPK, Akt, mTOR, p70S6K, LC3, BNIP3 and Beclin‐1. GAPDH was used as an internal control.

Figure 8

Detection of cell viability under autophagy regulation. (A) OC2 and (B) OCSL cells were treated with DMSO, 3‐MA, bafilomycin, rapamycin or honokiol, for 24 and 48 hr, and the cellular viability was measured by CCK‐8 analysis. The data present as the mean ± S.D. of three independent experiments.

Figure 9

Combinatory inhibition of honokiol and 5‐FU on cell proliferation. The cellular viability of (A) OC2 and (B) OCSL cells treated with DMSO, honokiol and 5‐FU for 24, 48 and 72 hr was measured by CCK‐8 analysis. The data present as the mean ± S.D. of three independent experiments.

Figure 10

Honokiol suppresses tumour progression in nude mice. (A) The cell viability and (B) cellular apoptosis of SAS cells were evaluated under honokiol treatment. (C) Subcutaneous tumours derived from SAS cells were treated with or without honokiol as described. Tumour volumes were evaluated as the mean of at least three mice per group. *: honokiol 5 mg/kg versus DMSO; #: honokiol 15 mg/kg versus DMSO. (D) Inhibitory effects of honokiol on xenograft tumour weight. *P < 0.05, **P < 0.01 compared with DMSO. (E) Haematoxylin and eosin staining in liver and kidney specimens of DMSO and honokiol‐treated mouse (Magnification, ×100 and ×200). (F) Apoptosis induction in tumour sections with TUNEL staining. The arrow indicates the colocalization of apoptosis‐induced nuclear DNA fragmentation. (Scale bar, 20 μm) (G) Autophagy elevation in tumour sections were determined by immunohistochemical staining (Magnification, ×200).