Meridianin C inhibits the growth of YD‐10B human tongue cancer cells through macropinocytosis and the down‐regulation of Dickkopf‐related protein‐3

Abstract Meridianin C is a marine natural product known for its anti‐cancer activity. At present, the anti‐tumour effects of meridianin C on oral squamous cell carcinoma are unknown. Here, we investigated the effect of meridianin C on the proliferation of four different human tongue cancer cells, YD‐8, YD‐10B, YD‐38 and HSC‐3. Among the cells tested, meridianin C most strongly reduced the growth of YD‐10B cells; the most aggressive and tumorigenic of the cell lines tested. Strikingly, meridianin C induced a significant accumulation of macropinosomes in the YD‐10B cells; confirmed by the microscopic and TEM analysis as well as the entry of FITC‐dextran, which was sensitive to the macropinocytosis inhibitor amiloride. SEM data also revealed abundant long and thin membrane extensions that resemble lamellipodia on the surface of YD‐10B cells treated with meridianin C, pointing out that meridianin C‐induced macropinosomes was the result of macropinocytosis. In addition, meridianin C reduced cellular levels of Dickkopf‐related protein‐3 (DKK‐3), a known negative regulator of macropinocytosis. A role for DKK‐3 in regulating macropinocytosis in the YD‐10B cells was confirmed by siRNA knockdown of endogenous DKK‐3, which led to a partial accumulation of vacuoles and a reduction in cell proliferation, and by exogenous DKK‐3 overexpression, which resulted in a considerable inhibition of the meridianin C‐induced vacuole formation and decrease in cell survival. In summary, this is the first study reporting meridianin C has novel anti‐proliferative effects via macropinocytosis in the highly tumorigenic YD‐10B cell line and the effects are mediated in part through down‐regulation of DKK‐3.


| INTRODUCTION
Oral squamous cell carcinoma (OSCC) is the most common malignant tumour of the lip, oral cavity and oropharynx. Moreover, OSCC can be severely disfiguring leading to a poor quality of life, including loss of general cognitive, social, emotional or physical functions as well as social relationships. 1 The primary treatment of OSCC is surgical removal, but radiation therapy and/or chemotherapy have emerged as alternative treatment options for OSCC. [2][3][4] Characteristically, OSCC cells at the late stage of malignancies are very resistant to cancer therapy-mediated apoptosis. Thus, there is an urgent need for understanding resistance mechanism and identifying new drugs or substances which offer therapeutic efficacy against OSCC.
Macropinocytosis is an endocytic pathway that leads to the formation of single membrane irregular vesicles (macropinosomes) by the internalization of large patches of the plasma membrane along with associated extracellular fluid and solutes. 5 Unlike the canonical and endocytic pathways that depend on coat proteins such as clathrin or caveolin, one of the distinctive hallmarks of macropinocytosis is the formation of expansive membrane ruffles within the plasma membrane. 6 This membrane ruffling is initiated by the rapid polymerization of branching of actin filaments. 7 Multiple proteins and signalling factors are involved in macropinocytosis, including the Rho superfamily of GTPases (Rac, Cdc42), lipid components (phosphoinositides, cholesterol, phosphatidylinositol phosphates), phospholipid kinases, receptor tyrosine kinases and phosphatases, SNX-5 and DKK-3. [8][9][10][11][12][13][14][15][16] There is also large body of evidence that now shows macropinocytosis can be specifically activated by extracellular stimuli, such as growth factors and natural or synthetic chemicals, and the activated macropinocytosis can have effects on cell survival/proliferation or cell death/growth inhibition on distinct cell types including cancer cells. 17,18 Meridianin C is one of the marine derived indole alkaloids (meridianin A-G) isolated from the South Atlantic tunicate Aplidium meridianum. 19,20 Previously, meridianin C, D or G analogues/derivatives have been shown to inhibit the proliferation of human breast (MCF-7) and cervix (HeLa) cancer and leukaemia (MV4-11) cells. [21][22][23] At present, neither the anti-tumour effect nor the mode of action of meridianin C in OSCC is known. In this study, we investigated the effect of meridianin C at 1 μM concentration on growth of four different human oral carcinoma cell lines (YD-8, YD-10B, YD-38 and HSC-3) that were originated from tongue (YD-8, YD-10B, HSC-3) or lower gingiva (YD-38). 24 It is worth noting that YD-8 and YD-38 cells have no tumorigenicity, but YD-10B cells are highly tumorigenic and invasive/metastatic. 25,26 In this article, we report for the first time that meridianin C strongly inhibits the growth of YD-10B OSCC cells through macropinocytosis, and its growth inhibitory and macropinocytosis inducing effects on the cells are mediated in part via the reduced expression of DKK-3. heat-inactivated foetal bovine serum (FBS), 100 units/mL penicillin and 100 μg/mL streptomycin and maintained at 37°C in a humidified condition of 95% air and 5% CO 2 . Meridianin C was synthesized as previously described 23
The cell count assay was performed in triplicate. Data are mean ± SEM of three independent experiments. Survival is expressed as a percentage of control. In some experiments, YD-10B cells (0.5 × 10 6 /2 mL/well) or normal HGFs-1) (0.4 × 10 6 /2 mL/well) were also plated in 6-well plates overnight. Cells were then treated with

| Transmission electron microscopy (TEM)
YD-10B cells (0.5 × 10 6 /2 mL/well) were seeded in 6-well plates the day before meridianin C treatment. Cells were then treated with 1 μM meridianin C for 8 h, while DMSO was used as control. DMSO or meridianin C-treated cells were fixed overnight at 4°C with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4. Samples were post-fixed for 30 min with 0.5% osmium tetroxide/0.8% potassium ferricyanide, transferred to 1% tannic acid for 1 h and then to 1% uranyl acetate overnight at 4°C. Next, the samples were dehydrated with a graded ethanol series and embedded in Spurr's resin.
Thin sections were cut with a Leica EM UC6 ultramicrotome (Leica), and stained with 1% uranyl acetate and Reynold's lead citrate prior to viewing at 120 kV on a Tecnai BT Spirit transmission electron microscope (FEI). Digital images were acquired with a Hammamatsu XR-100 side mount digital camera system (Advanced Microscopy Techniques) and processed using Adobe Photoshop CS5 (Adobe Systems Inc.).

| Scanning electron microscopy
YD-10B cells were seeded in 6-well plates (0.5 × 10 6 cells/2 mL/ well) the day before meridianin C treatment. Cells were then treated with vehicle control (DMSO) or 1 μM meridianin C for 2 h.
The conditioned cells were centrifuged, fixed in 0.5% glutaraldehyde and 0.5% paraformaldehyde fixative, washed with 0.1 M phosphate-buffered saline (PBS), and post-fixed with 1% osmium tetroxide solution for 1 h. 2% tannic acid was used to conductively stain for 12 h, later washed with PBS, and fixed with 1% osmium tetroxide solution for 1 h. The specimens were dehydrated with ethanol, t-butyl alcohol and freeze dryer. Finally, the specimen was sputter coated with platinum-palladium (Pt-Pd) in an ion coater for 2 min, followed by microscopic examinations (S-4200, Hitachi Co., Tokyo, Japan).

| Measurement of DNA fragmentation
YD-10B cells were seeded in 6-well plates at a density of 0.5 × 10 6 cells per well the day before treatment. Cells were incubated with meridianin C or vehicle control (DMSO) for 4 to 48 h, at which point, cells were harvested, washed and lysed in a buffer containing 50 mM Tris (pH 8.0), 0.5% sarkosyl, 0.5 mg/mL proteinase K and 1 mM EDTA at 55°C for 3 h, followed by addition of RNase A (0.5 μg/mL) for a further 18 h at 55°C. The lysates were then cen-

| Fluorescein isothiocyanate (FITC) staining
To monitor the functionality of meridianin C-induced macropinocytosis (macropinosome formation/internalization), 0.25 × 10 5 YD-10B cells/mL were seeded on coverslips and treated with meridianin C (1 μM) and/or FITC-dextran (0.5 mg/mL) in the presence or absence of amiloride (4 mM) for 4 h. The cells were washed twice with PBS and mounted onto microscopic glass slides using Permafluor aqueous mounting media (Thermo Scientific, Waltham, MA, USA) media.
Bright field and fluorescence were observed using a Zeiss AxioObserver.A1 inverted microscope (Carl Zeiss, Germany) and images acquired using Zen 2 software (Carl Zeiss). Fluorescent intensity was quantified using Image-J software.

| Preparation of whole cell lysates
To see the effect of meridianin C on expression of apoptosis-or macropinocytosis-related proteins, YD-10B cells (0.5 × 10 6 /2 mL/ well) were seeded in 6-well plates the day before meridianin C treatment. Cells were treated with meridianin C (1 μM) or vehicle control (DMSO) for the indicated times. At each time-point, cells were washed twice with PBS and proteins extracted using a modified RIPA buffer (50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 0.1% sodium dodecyl sulphate, 0.25% sodium deoxycholate, 1% Triton X-100, 1% PARK ET AL.
The supernatants were saved and protein concentrations determined by bicinchoninic acid assay (BCA) protein assay (Pierce).

| Statistical analyses
Cell count analysis was performed in triplicates and repeated three times. Data were expressed as mean ± SEM. The significance of difference was determined by one-way ANOVA (Laerd Statistics, Chicago, IL, USA). All significance testing was based upon a P < 0.05.

| Meridianin C has strong growth inhibitory and vacuole-inducing effects on YD-10B cells
We investigated the treatment effect of meridianin C ( Figure 1A

| Meridianin C-induced macropinocytosis (macropinosome formation/internalization) in YD-10B cells is functional
Amiloride inhibits macropinocytosis by blocking the activity of Na/H exchanger, causing a decrease in pH at the plasma membrane. 30 This amiloride-induced pH drop suppresses the membrane ruffling and membrane extensions that form the macropinosome. 31 Soluble sugars or proteins, such as dextran or albumin, are useful fluorescent markers specific for macropinocytosis. 32 Using amiloride and fluorescein isothiocyanate-dextran with molecular weight of 70 kDa (FITCdextran), we sought to explore whether the meridianin C-induced macropinocytosis (macropinosome formation/internalization) in YD-10B cells is functional. Microscopic observations clearly demonstrated that amiloride strongly blocked the meridianin C-induced accumulation of vacuoles in YD-10B cells ( Figure 4A). Moreover, meridianin C increased the delivery of FITC-dextran into YD-10B cells while amiloride strongly inhibited it ( Figure 4B). Further, fluorescence intensity of FITC-dextran (green colour) was determined by Image-J software ( Figure 4C).

DKK-3 protein in YD-10B cells
DKK-3 plays a role in modulating macropinocytosis in T24 human bladder cancer cells 16 and is also expressed in OSCC-derived cell lines. 33 We therefore sought to explore whether DKK-3 is expressed in YD-10B cells and is regulated by meridianin C treatment. As shown in Figure 5A, high cellular levels of DKK-3 protein were seen in YD-10B cells, and its expression level was even higher relative to β-actin than that in T24 cells. There was no detectable DKK

| DKK-3 knock-down led to partial accumulation of vacuoles in YD-10B cells along with partial decrease of the cell growth
To evaluate the role of reduced DKK-3 in meridianin C's growth inhibition and/or macropinocytosis in YD-10B cells, we transfected con-  Figure 6B) accompanied with a partial but significant reduction in cell survival ( Figure 6C).

| Overexpression of DKK-3 largely blocks meridianin C-induced accumulation of vacuoles in YD-10B cells along with significant increase of the cell survival
To further clarify that DKK-3 plays an important role in the function of meridianin C, we next performed DKK-3-Flag cDNA transfection experiments to generate stable YD-10B cells that overexpress DKK-3-Flag. Using these stable clones, we tested whether DKK-3 overexpression could interfere with the meridianin C's growth inhibitory and vacuole-inducing activities in YD-10B cells. As shown in Figure 7A Importantly, while meridianin C treatment largely induced many vacuoles in YD-10B mock cells, it had no effect on the formation of vacuoles in YD-10B DKK-3 cells ( Figure 7B). Furthermore, as shown in Figure 7C, the meridianin C-induced reduction in YD-10B cell survival was considerably blocked by DKK-3 overexpression.

| Comparison of the effects of meridianin C and its derivatives on growth and macropinosome (vacuole) accumulation in YD-10B cells
We have recently synthesized a novel series of meridianin C derivatives substituted at C-5 position (7a-j) and reported their anti- speculate that meridianin C's growth inhibitory and/or macropinocytosis inducing effects on YD-10B cells may be through acting upon one or more of these kinases, which yet remain to be elucidated.
A notable finding of the present study is meridianin C regulation of DKK-3 expression in YD-10B cells. DKK-3 is one of the DKK protein families and is known as a Wnt antagonist. 41,42 It is also recognized as a potential tumour suppressor, based on the facts that DKK-3 expression is frequently down-regulated in a wide array of malignancies. 43,44 Recent evidence indicates that DKK-3 may also play additional roles in cancer cell survival. DKK-3 knock-down by siRNA in T24 human bladder cancer cells inhibited cell growth and induced macropinocytosis and autophagy. 16 It also has been recently demonstrated that DKK-3 is expressed in a subset of OSCC-derived cell lines including HSC-4. 33 Moreover, in HSC-4 cells, DKK-3 knock-down inhibited migration and invasion.
These results imply that DKK-3 may have a metastasis-related oncogenic function in OSCC. Here we found DKK-3 is highly expressed in YD-10B cells and meridianin C suppresses its expression. To the best of our knowledge, this is the first study showing the ability of meridianin C to down-regulate DKK-3 in cancer cells.
Of further importance, the present study revealed that siRNA- to elucidate its mechanism of action.
We have recently synthesized a series of meridianin C derivatives, and demonstrated their anti-proliferative activity in human leukaemia cell lines including MV4-11. 23 In the current study, we further compared the growth inhibitory and/or macropinocytosis inducing effects of meridianin C and its derivatives on YD-10B cells.
Although some of meridianin C derivatives had macropinocytosis inducing and growth inhibitory effects on YD-10 cells, it was evident that their effects were much weaker than those induced by its parental compound meridianin C. These results indicate that meridianin C has a unique structural moiety leading to strong growth suppressive and macropinocytosis inducing activity in YD-10B cells, which is distinct from other cancer cells.
In summary, meridianin C induces a significant level of macropinocytosis and selectively decreases the survival of YD-10B cells in part through a reduction in DKK-3. Although there are still important issues that remain to be resolved, including meridianin C's macropinocytosis inducing and anti-tumour effects on animal models, our findings suggest that meridianin C is a potential novel target for metastatic human tongue cancer.