**5. Tubulin bundle was formed by docetaxel treatment**

Taxanes, including docetaxel, function as a mitotic spindle toxin by inhibiting microtubule turnover. They bind to microtubules and enhance tubulin polymerization. We hypothesized that docetaxel may exert similar effect on cytosolic, non-centrisome associated microtubules resulting in decreased cell motility. We therefore examined the structure of microtubules as well as actin filaments. Consistent with previous observation, filopodia formation was less in docetaxel treatment compared to cisplatin treatment. But no gross abnormality was found in actin filament structure between the treatments. On the other hand, tubulin bundle formation was noted in docetaxel treatment but not in cisplatin treatment (Fig.3.). Then, we attempted to find the mechanism that connect deformed microtubule and decreased filopodia formation.

Fig. 3. Staining of α-tubulin (by antibody) in HEp-2 cells treated by cisplatin, docetaxel or no treatment at IC50. Bars=50 µm. 3 replicate were used in each experiments and experiments were repeated 4 times.

Microtubule and Cdc42 are the Main Targets of

were repeated 4 times. Arrows; filopodia

treated with cisplatin or docetaxel.

experiments were repeated 3 times.

cytoskeleton remodeling.

Docetaxel's Suppression of Invasiveness of Head and Neck Cancer Cells 155

treatment, either. These results suggest that docetaxel treatment does not directly affect actin

Fig. 5. Staining of F-actin (phalloidin) in HEp-2 cells treated by cisplatin, docetaxel or no treatment at IC50. Bars=50 µm. 3 replicate were used in each experiments and experiments

Fig. 6. Time course of the levels of p-ERM, cofilin and LIMK in HEp-2 cells and Ca9-22 cells

β-actin was probed for loding control. 3 replicate were used in each experiments and

#### **6. Docetaxel inhibited Cdc42 activity**

Rho GTPases regulate many essential cellular processes, including actin dynamics, gene transcription, cell-cycle progression, cell adhesion, tumor progression and invasiveness (Hall 1998; Schmitz et al. 2000; Price et al. 2001). Among Rho-GTPases, Cdc42 is previously implicated in connecting microtubular input to actin filament organization (Cau et al. 2005). Cdc42 also promotes leading-edge extension through activation of Rac, which is implicated in formation of lamellipodia (Bishop et al. 2000). Thus, we examined activity of Cdc42, Rac and RhoA in the cells underwent cisplatin or docetaxel treatment, or no treatment. At IC10 concentration, docetaxel significantly decreased Cdc42 and Rac activity in HEp-2 cells, but not RhoA activity (Fig.4.; cited from (Kogashiwa et al. 2010) with modification). Total amount of Cdc42, Rac and RhoA was not significantly different among these three conditions.

It is reported that Cdc42 is activated in a thin band at cell edges extending filopodia (Nalbant et al. 2004). Consistent with the results of activity assay, Cdc42 localized at the plasma membrane was decreased after docetaxel treatment at IC10. Localization of Rac1 and RhoA had no apparent changes after treatment compared to control.

Fig. 4. Colorimetric assay of Cdc42, Rac and RhoA activity in HEp-2 cells. The levels of activated Cdc42, Rac and RhoA in HEp-2 cell were evaluated immediately after 1 hr of indicated treatment. Each data point represents mean ± SE. \*p < 0.05, \*\*p < 0.01.

#### **7. The molecules implicated in actin cytoskelton regulation were not significantly different between cisplatin and docetaxel treatment.**

Lamellipodia or filopodia formation was suppressed when cells were treated with docetaxel (fig.5.). Ezrin/radixin/moesin (ERM) proteins link the cortical cytoskeleton to the plasma membrane. In their active conformation (i.e. phosphorylated ERM), the N-terminal ERM domain binds to the cytoplasmic tails of transmembrane proteins, and the C-terminal ERM association domain binds to actin filaments. Using a p-ERM antibody, the levels of active ERM proteins were evaluated by Western blotting after no treatment, cisplatin or docetaxel treatment at IC10. There was no significant difference in the level of p-ERM among cisplatin, docetaxel and no treatment over a time course up to 48 hours (fig.6.). We also investigated the cofilin pathway as a regulator of the actin cytoskeleton. Cofilin is able to bind both Gactin and F-actin, and regulated by LIM kinase 1 and its related kinases. The levels of cofilin (fig.6.) and LIMK1 (fig.6.) were evaluated over a time course of treatment at IC10. The levels of these proteins were not significantly different among cisplatin, docetaxel and no

Rho GTPases regulate many essential cellular processes, including actin dynamics, gene transcription, cell-cycle progression, cell adhesion, tumor progression and invasiveness (Hall 1998; Schmitz et al. 2000; Price et al. 2001). Among Rho-GTPases, Cdc42 is previously implicated in connecting microtubular input to actin filament organization (Cau et al. 2005). Cdc42 also promotes leading-edge extension through activation of Rac, which is implicated in formation of lamellipodia (Bishop et al. 2000). Thus, we examined activity of Cdc42, Rac and RhoA in the cells underwent cisplatin or docetaxel treatment, or no treatment. At IC10 concentration, docetaxel significantly decreased Cdc42 and Rac activity in HEp-2 cells, but not RhoA activity (Fig.4.; cited from (Kogashiwa et al. 2010) with modification). Total amount of Cdc42, Rac and RhoA was not significantly different among these three

It is reported that Cdc42 is activated in a thin band at cell edges extending filopodia (Nalbant et al. 2004). Consistent with the results of activity assay, Cdc42 localized at the plasma membrane was decreased after docetaxel treatment at IC10. Localization of Rac1 and

The levels of activated Cdc42, Rac and RhoA in HEp-2 cell were evaluated immediately after 1 hr of indicated treatment. Each data point represents mean ± SE. \*p < 0.05, \*\*p < 0.01.

Lamellipodia or filopodia formation was suppressed when cells were treated with docetaxel (fig.5.). Ezrin/radixin/moesin (ERM) proteins link the cortical cytoskeleton to the plasma membrane. In their active conformation (i.e. phosphorylated ERM), the N-terminal ERM domain binds to the cytoplasmic tails of transmembrane proteins, and the C-terminal ERM association domain binds to actin filaments. Using a p-ERM antibody, the levels of active ERM proteins were evaluated by Western blotting after no treatment, cisplatin or docetaxel treatment at IC10. There was no significant difference in the level of p-ERM among cisplatin, docetaxel and no treatment over a time course up to 48 hours (fig.6.). We also investigated the cofilin pathway as a regulator of the actin cytoskeleton. Cofilin is able to bind both Gactin and F-actin, and regulated by LIM kinase 1 and its related kinases. The levels of cofilin (fig.6.) and LIMK1 (fig.6.) were evaluated over a time course of treatment at IC10. The levels of these proteins were not significantly different among cisplatin, docetaxel and no

RhoA had no apparent changes after treatment compared to control.

Fig. 4. Colorimetric assay of Cdc42, Rac and RhoA activity in HEp-2 cells.

**7. The molecules implicated in actin cytoskelton regulation were not significantly different between cisplatin and docetaxel treatment.** 

**6. Docetaxel inhibited Cdc42 activity** 

conditions.

treatment, either. These results suggest that docetaxel treatment does not directly affect actin cytoskeleton remodeling.

Fig. 5. Staining of F-actin (phalloidin) in HEp-2 cells treated by cisplatin, docetaxel or no treatment at IC50. Bars=50 µm. 3 replicate were used in each experiments and experiments were repeated 4 times. Arrows; filopodia

Fig. 6. Time course of the levels of p-ERM, cofilin and LIMK in HEp-2 cells and Ca9-22 cells treated with cisplatin or docetaxel.

β-actin was probed for loding control. 3 replicate were used in each experiments and experiments were repeated 3 times.

Microtubule and Cdc42 are the Main Targets of

**by cisplatin and docetaxel treatment** 

treatment (Figure 8).

Fig. 8. Gelatin zaymograpy.

**10. Discussion** 

unchanged by either cisplatin or docetaxel treatment

Docetaxel's Suppression of Invasiveness of Head and Neck Cancer Cells 157

**9. Matrix metalloproteinase (MMP) production was not significantly affected** 

MMPs are known to play an important role in extracellular matrix remodeling during the process of tumor invasion and metastasis (Egeblad et al. 2002). Two of these enzymes, MMP-2 and MMP-9, are potent gelatinases and have been correlated with the processes of invasion and metastasis of SCC (Sheu et al. 2003; Patel et al. 2005). Gelatin zymography revealed prominent 72000 dalton bands, corresponding to MMP2 secreted from the HEp-2 and Ca9-22 cells. These bands appeared unchanged by either cisplatin or docetaxel

MMP secretion in HEp-2 cells and Ca9-22 cells after treatment or control were evaluated by

The metastatic process has traditionally been viewed as follows: (1) detachment of individual cells from the primary lesion (2) invasion of local stroma (3) entry of single cells or aggregates of tumor cells into blood vessels directly or via lymphatic channels (intravasation) (4) sticking to the vasculature distant from their origin followed by extravasation, and (5) invasion into the parenchyma of the target organ site. The newly formed lesions can themselves become the source of disseminating cells which repeat this cycle, giving rise to tertiary metastasis. Thus, Inhibition of invasion in the primary lesion

Gelatin zymography. Gelatin zymography revealed prominent 72000 dalton bands, corresponding to MMP2 secreted from the HEp-2 and Ca9-22 cells. These bands appeared

#### **8. Docetaxel treatment did not promote epithelial-mesenchymal transition (EMT)**

It has been well documented that many cancer cells lose most of their epithelial characteristics during progression and metastasis, through the process of EMT (Thiery 2002). Generally, EMT causes increased motility and invasiveness of cancer cells due to decreased cell-cell adhesion. Snail, a zinc finger transcription factor, triggers EMT through direct repression of E-cadherin transcription (Batlle et al. 2000; Cano et al. 2000). The reverse correlation of snail and E-cadherin expression has been reported for various human cancers, including SCC (Yokoyama et al. 2001). Accordingly, we investigated the snail and Ecadherin expression levels to assess whether cisplatin and/or docetaxel at IC10 differently influences EMT. Snail was decreasing over a time course (Fig.7.). Conversely E-cadherin was increasing over a time course (Fig.7.). But the levels of these proteins were not significantly different between cisplatin, docetaxel and no treatment. These results indicate that docetaxel treatment does not promote EMT at least in these cell lines.

Fig. 7. Time course of snail and E-cadherin expression of HEp-2 cells and Ca9-22 cells treated with IC10 concentration of cisplatin or docetaxel.

β-actin was probed for loding control. 3 replicate were used in each experiments and experiments were repeated 3 times.
