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SB-T-121205, a next-generation taxane, enhances apoptosis and inhibits migration/invasion in MCF-7/PTX cells

Breast cancer is the leading cause of cancer death among women. Paclitaxel, a mitotic inhibitor, is highly effective in the treatment of breast cancer. However, development of resistance to paclitaxel limits its clinical use. Identifying new compounds and new strategies that are effective against breast cancer, in particular drug-resistant cancer, is of great importance. The aim of the present study was to explore the potential of a next-generation taxoid, SB-T-121205, in modulating the proliferation, migration and invasion of paclitaxel-resistant human breast cancer cells (MCF-7/PTX) and further evaluate the underlying molecular mechanisms. The results of MTT assay showed that SB-T-121205 has much higher potency to human breast cancer cells (MCF-7/S, MCF-7/PTX and MDA-MB-453 cells) than paclitaxel, while that the non-tumorigenic human bronchial epithelial cells (BEAS-2B) were slightly less sensitive to SB-T-121205 than paclitaxel. Flow cytometry and western blot methods revealed that SB-T-121205 induced cell cycle arrest at the G2/M phase and apoptosis in MCF-7/PTX cells through accelerating mitochondrial apoptotic pathway, resulting in reduction of Bcl-2/Bax ratio, as well as elevation of caspase-3, caspase-9, and poly(ADP-ribose) polymerase (PARP) levels. Moreover, SB-T-121205 changed epithelial-mesenchymal transition (EMT) property, and suppressed migration and invasion abilities of MCF-7/PTX cells. Additionally, SB-T-121205 exerted antitumor activity by inhibiting the transgelin 2 and PI3K/Akt pathway. These findings indicate that SB-T-121205 is a potent antitumor agent that promotes apoptosis and also recedes migration/invasion abilities of MCF-7/PTX cells by restraining the activity of transgelin 2 and PI3K/Akt, as well as mitochondrial apoptotic pathway. Such results suggest a potential clinical value of SB-T-121205 in breast cancer treatment.


Introduction
Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer-related mortality among females worldwide (1). Currently, standard therapies for breast cancer patients include surgery, radiotherapy and chemotherapy, which plays an irreplaceable role (2). Paclitaxel (PTX; Fig. 1A), a first-line therapeutic agent used clinically to treat breast cancer, exerts its antitumor activity by promoting the polymerization of tubulin and stabilizing the resulting microtubules, causing cell cycle arrest at the G2/M phase that leads to apoptotic death of cancer cells (3). However, paclitaxel resistance often occurs after a period of treatment, causing a serious problem in chemotherapy (4). Accumulated studies manifest that there are several major mechanisms for paclitaxel resistance, including point mutations in β-tubulin, alterations in the expression of β-tubulin isotypes, particularly the β-Ⅲ tubulin isoform, overexpression of ATP-binding cassette transporters and suppression of apoptosis. More importantly, the drug-resistance to chemotherapy may eventually result in mortality of cancer patients due to tumor metastasis (5,6). It is therefore highly desirable to develop novel agents with

SB-T-121205, a next-generation taxane, enhances apoptosis and inhibits migration/invasion in MCF-7/PTX cells
minimum side-effects and improved activity against various tumors, especially against drug-resistant human breast cancer. In the last decades, a number of taxanes have been designed and synthesized based on the structure of the first-generation taxanes, paclitaxel and docetaxel. Many of these new taxanes exhibited strong antitumor activity against paclitaxel-resistant tumor xenografts. For instance, Roh et al (7) found two of the 3'-N-acyl-paclitaxel analogues, in which the phenyl group of 3'-N-benzoyl was replaced with 1-cyclopentenyl (1k) and 1-cyclohexenyl (1n), displayed seven times higher cytotoxicity than paclitaxel against doxorubicin-resistant breast cancer cells. It was also reported that another taxane, Lx2-32c, was active against paclitaxel-resistant breast cancer cells (MX-1/T) through intrinsic apoptosis signaling pathway, and exhibited efficacy against its tumor xenografts in nude mice, which suggests the potential of Lx2-32c to be a promising drug candidate (8). TPI-287, a new microtubule stabilizer, showed cytotoxicity similar to that of paclitaxel in breast cancer cells and efficacy against primary breast tumor xenografts in an animal model. Also, it was found to significantly decrease metastatic colonization of breast cancer in the brain (9). Moreover, Ojima et al (10) developed a series of novel second-generation taxanes with systematic modifications at the C2, C10, C3' and C3'N positions. For example, among these new-generation taxanes synthesized and assayed, SB-T-1214 and SB-T-121303, exhibited significantly lower IC 50 values, 9.00±0.77 nM and 3.65±0.21 nM, respectively for paclitaxel-resistant ovarian cancer cells than paclitaxel (532.95±3.18 nM). Such results clearly warrant further exploitation of next-generation taxanes with superior potency, efficacy and pharmacological properties against breast cancer.
Transgelin 2 is reported to be implicated in tumorigenesis, boosting tumor progression and promoting metastases (11). Additionally, abnormal expression of transgelin 2 was discovered in lung, gastric and colorectal cancer (12)(13)(14). We previously reported that transgelin 2 expression was extremely high in paclitaxel-resistant human breast cancer cells (MCF-7/PTX) compared to breast cancer drug-sensitive cells by proteomics analysis (15). Knockdown of transgelin 2 via small interfering RNA sensitized MCF-7/PTX cells to paclitaxel, and suppressed their migration/invasion abilities, suggesting that transgelin 2 might be a new biomarker for breast cancer (16). On the other hand, aberrant activation of the phosphatidylinositol 3 kinase/serine-threonine kinase (PI3K/Akt) pathway contributes to chemo-resistance, tumor metastasis and poor prognosis (17,18). Notably, we reported that the PI3K/Akt pathway was activated in MCF-7/PTX cells and the TAGLN2-knockdown inhibited the PI3K/Akt pathway, suggesting that the PI3K/Akt pathway would be critical to breast cancer progression (19).
We confirmed that the MCF-7/PTX cells, developed by our laboratory for the assay used, were highly resistant to paclitaxel and exhibited strong migration/invasion capacities (20,21). In the present study, eight novel next-generation taxanes were screened by MTT assay. Among these taxanes, SB-T-121205 ( Fig. 1B) was found to be highly potent against the paclitaxel-resistant MCF-7/PTX cells. Subsequently, the effect of SB-T-121205 on cell apoptosis, epithelial-mesenchymal transition (EMT) property, migration and invasion was assessed in MCF-7/PTX cells, along with the underlying molecular mechanisms. Our data indicated that SB-T-121205 exerted its high potency against MCF-7/PTX cells through activation of the transgelin 2 and PI3K/Akt pathway, which suggests that SB-T-121205 would serve as an efficacious drug candidate for breast cancer treatment.
Cell lines and cell culture. The human breast cancer cell lines, MCF-7/S and MDA-MB-453, and non-tumorigenic human bronchial epithelial cell line (BEAS-2B) were obtained from the Cell Bank of Shanghai, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. MCF-7/PTX cell line was successfully established as previously described (16) with the concentration of 30 nM paclitaxel. The cells were grown in RPMI-1640 medium (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin (Harbin Pharmaceutical Group, Co., Ltd., Harbin, China)/streptomycin (North China Pharmaceutical Group, Co., Ltd., Shijiazhuang, China) at 37˚C in a humidified atmosphere of 5% CO 2 . Cells in exponential phase growth were observed under inverted light microscope (Olympus Corp., Tokyo, Japan).
MTT assay. Cells at 5x10 5 /ml density were seeded into 96-well plates (Corning, Inc., Corning, NY, USA) with 100 µl medium for the duration indicated. After 72 h, 20 µl of MTT (5 mg/ml) was added into each well and incubated at 37˚C for 4 h. Then, 150 µl of dimethyl sulfoxide (DMSO) was added into each well for dissolving the formazan for 15 min. The absorbance was tested at 490 nm on a microplate reader (BioTek Instruments, Inc., Winooski, vT, USA). The 50% growth inhibitory concentration (IC 50 ) of drug was calculated to evaluate the drug sensitivity. Each experiment was repeated three times.
Flow cytometry assay. For cell cycle assay, cells were exposed to paclitaxel (600 nM) or SB-T-121205 (10 or 20 nM) for 48 h and then harvested. After three washes with cold phosphatebuffered saline (PBS), cells were fixed with 70% cold ethanol at 4˚C overnight. The next day, cells were washed with cold PBS and suspended in a 500 µl staining solution of propidium iodide (PI; Sigma-Aldric) staining solution at 37˚C for 30 min without light. The samples were tested using FACSCanto™ II flow cytometry (Becton Dickinson, Franklin Lakes, NJ, USA).
For cell apoptosis analysis, cells with different treatments after 48 h were collected, washed and were resuspended with cold PBS. Then, the cells were double stained with Annexin V-FITC and PI at 37˚C for 20 min from light with an Annexin-v-FLUOS staining kit in accordance with the manufacturer's instructions. The stained cells were analyzed using FACSCanto™ II flow cytometry. Annexin V + /PIwere regarded as early apoptotic cells and Annexin v + /PI + were late apoptotic cells. Experiments were repeated in triplicate, independently.
Wound healing scratch assay. Cells (5x10 5 /ml) were seeded in 6-well plate until confluent. Cells were serum-starved overnight and an artificial scratch wound was created. The cells were then maintained in serum-free culture at 37˚C in a humidified atmosphere of 5% CO 2 . Migration photos were captured at 0, 24 and 48 h after scratching. Experiments were repeated in triplicate independently. Percent wound closure was calculated using the following equation: percent wound Transwell invasion assay. The invasiveness of cells was evaluated by a Boyden chamber method. The polycarbonate filters (8 µm pore size; Corning) were coated with Matrigel Matrix (BD Biosciences, San Jose, CA, USA) and incubated at 37˚C for 5 h. Next, 5x10 5 cells suspended in 200 µl serumfree RPMI-1640 were added into the upper chamber, while 800 µl of complete media was added to the lower chamber. After 48 h, cells migrated through Matrigel and adhered onto the lower chamber was fixed in 4% paraformaldehyde for 30 min, stained with 0.1% crystal violet (Beyotime Institute of Biotechnology) and counted by fluorescent microscopy (Olympus). Each invasion assay was repeated in three independent experiments.
Statistical analysis. Statistical analysis was performed using one-way ANOvA. All values were expressed as mean ± standard deviation (SD) from triplicate experiments performed in a parallel manner. P<0.05 were considered statistically significant.

Results
The intrinsic cytotoxicity of SB-T-121205 on cancer and normal cells. The intrinsic cytotoxicity of the eight novel taxanes against MCF-7/PTX cells were measured by MTT assay. Treating cells with these taxanes for 72 h significantly inhibited cell growth. Among these taxanes examined, SB-T-121205 showed the highest inhibition. We therefore, chose SB-T-121205 for further study. After 72 h of exposure, SB-T-121205 obviously restrained growth of MCF-7/PTX cells with an IC 50 value of 19.01±2.03 nM, which was two orders of magnitude lower than paclitaxel (2290.87±125.18 nM) (16) ( Table I and Fig. 1C and D). The molecular formula of  (Fig. 1C and D). We examined the cell growth inhibitory effect of SB-T-121205 on MDA-MB-453 cells. SB-T-121205 was found to have slightly lower IC 50 value (38.67±3.58 nM) than paclitaxel (IC 50 54.62±3.28 nM) (Fig. 1E). We also examined the cytotoxicity of paclitaxel and SB-T-121205 against non-tumorigenic BEAS-2B human bronchial epithelial cells. Treating BEAS-2B cells with different concentration of paclitaxel or SB-T-121205 revealed that BEAS-2B cells are slightly less sensitive to SB-T-121205 (IC 50 59.80±1.89 nM) than paclitaxel (IC 50 54.57±2.10 nM) (Fig. 1F). Thus, for the treatment of MCF7/S and MCF7/PTX and MDA-MB-453 cancer cells, SB-T-121205 has much wider therapeutic index than paclitaxel.

SB-T-121205 induces apoptosis and G2/M arrest in MCF-7/PTX cells.
To confirm whether SB-T-121205 triggered apoptosis of MCF-7/PTX cells, we carried out Annexin v and PI double staining followed by flow cytometric analysis. MCF-7/PTX cells were incubated with paclitaxel alone or SB-T-121205 alone for 48 h. As shown in Fig 2A, paclitaxel (600 nM) and SB-T-121205 (10 or 20 nM) indeed increased cell apoptosis rates that reached 33.48, 31.78 and 39.81%, respectively ( Fig. 2A). Since the concentration used for paclitaxel is 30-60 times higher than that of SB-T-121205, SB-T-121205 is far better than paclitaxel in promoting cell apoptosis. In brief, it is confirmed that SB-T-121205 has the ability to induce apoptosis in MCF-7/PTX cells.
To further investigate the mechanisms that SB-T-121205 inhibits the cancer cell growth, the MCF-7/PTX cells were exposed to various concentrations of SB-T-121205 for 48 h, and then cell cycle analysis was performed.  (Fig. 2B). The results confirmed that SB-T-121205 arrests the mitosis of MCF-7/PTX cells at

SB-T-121205 suppresses the EMT property of MCF-7/PTX cells.
It is well known that EMT is a critical event in the development of cancers (22). To further examine whether SB-T-121205 could alter the EMT property of MCF-7/PTX cells, mammosphere formation and western blot assays were performed. As Fig. 3A shows, SB-T-121205 exposure triggered morphological changes in the MCF-7/PTX cells from elongated shape to cobblestone shape (Fig. 3A), which is a characteristic EMT morphology. After treated with SB-T-121205, the mammosphere forming ability of MCF-7/PTX cells was decreased (Fig. 3B). In addition, a low concentration (20 nM) of SB-T-121205 distinctly increased the expression of epithelial marker E-cadherin, whereas the levels of mesenchymal markers N-cadherin and vimentin were reduced (Fig. 3C). However, the effect of high concentration (600 nM) of paclitaxel was inferior to that of the low concentration of SB-T-121205. Thus, SB-T-121205 possesses much higher potency than paclitaxel for suppression of EMT in MCF-7/PTX cells.   (Fig. 5A and  B). SB-T-121205 exhibited its effects on migration and invasion of MDA-MB-435 cells in a dose-dependent manner, and was clearly more effective than paclitaxel. Thus, we have found that SB-T-121205 not only suppresses the EMT process, but also inhibits migratory and invasive abilities of MCF-7/PTX and MDA-MB-435 human breast cancer cells much more effectively than paclitaxel.

SB-T-121205 represses transgelin 2 expression and activation of the PI3K/Akt pathway in MCF-7/PTX cells.
To understand molecular mechanism by which SB-T-121205 receded cell proliferation and migratory/invasive abilities of MCF-7/PTX cells, the protein levels of transgelin 2 and the PI3K/Akt pathway were analyzed. After the treatment of MCF-7/ PTX cells with SB-T-121205, the expression of transgelin 2, p-Akt and p-GSK-3β was downregulated with a concomitant augment expression of PTEN, whereas the levels of Akt and GSK-3β were unaffected (Fig. 6A).
Furthermore, the deactivation of Akt signaling pathway augmented the expression of pro-apoptotic factor Bax and   the cleavage of caspase-3, caspase-9, PARP significantly, while suppressed pro-survival factor Bcl-2, ultimately leading to apoptosis. On the other hand, the downstream molecule Snail was strongly downregulated (Fig. 6B). In short, these observations suggest that SB-T-121205 is an antitumor agent that affects cell behavior by modulating transgelin 2 and the PI3K/Akt pathway.

Discussion
Paclitaxel was first approved in April 1992 for the treatment of platinum-resistant breast cancer by the Food and Drug Administration, USA. It exerts vital anticancer effect through its distinct antimitotic mechanism of action (24). However, a number of studies since then have revealed that treatment  with paclitaxel brought about undesirable adverse effects, including drug-resistance, hence seriously restricting its therapeutic effects and clinical applications (25). For this reason, it is essential to deal with the problem of paclitaxel resistance to enhance the survival of patients with breast cancer.
There are two main ways to solve the problem of drug resistance. The first is to develop resistance reversal agents. Scientists in the past few decades have screened many compounds to identify compounds with multidrug resistance reversal activities against various drug-resistant cell lines. Compounds such as verapamil (26) and metformin (27) were discovered through these efforts and developed. These compounds sensitize tumor cells to chemotherapeutic drugs, and this line of research is continuing. The second is to discover novel compounds with antitumor activities that may have high efficacy against drug-resistant targets and then directly kill cancer cells, which would also provide new insights into prevention and treatment of breast cancer.
Considerable efforts have been made to develop newgeneration taxoids with outstanding antitumor activity, much better than paclitaxel. For example, Nikolakakis et al (28) reported that 7β-O-glycosylated taxanes (9 and 15) were more potent (IC 50 12-15 nM) than docetaxel (IC 50 40 nM) and much more potent than paclitaxel (IC 50 >5 µM) against adriamycin-resistant human breast cancer cells (MCF-7/ADR). It was also documented that 2'-(N-methylpyridinium acetate) derivative of paclitaxel showed excellent potency in lung cancer cells and breast cancer cells (29). Based on structureactivity relationships (SAR) study, a series of new-generation taxanes have been developed by Ojima et al (10,30), which exhibited 2-3 orders of magnitude higher potency than paclitaxel or docetaxel against multidrug-resistant breast, ovarian, colon, pancreatic and prostate cancer cell lines (31). These new-generation taxanes have modifications at C10, C3', C3'N and/or C2. A newly developed next-generation taxane, SB-T-121205 possesses a 3-trifluoromethoxylbenzoyl group at C2 on the top of modifications in the new-generation taxanes mentioned above. The present study disclosed, for the first time, the excellent activities of SB-T-121205 in inhibiting the growth of MCF-7/S, MCF-7/PTX and MDA-MB-453 human breast cancer cells. An interesting observation in this study was that BEAS-2B normal human cells were relatively insensitive to SB-T-121205, which means that SB-T-121205 has a good therapeutic index. It was observed that the apoptosis induced at 20 nM SB-T-121205 in MCF-7/PTX cells was more powerful than 600 nM paclitaxel, suggesting SB-T-121205 possesses an extremely strong anti-proliferative activity. SB-T-121205 induced G2/M phase arrest in MCF-7/PTX cells in a manner similar to paclitaxel. In addition, SB-T-121205 changed cell morphology, modulated EMT marker expression and weakened the mammosphere forming ability, then mitigated the EMT process in MCF-7/ PTX cells. Importantly, SB-T-121205 exhibited its ability to restrain the migration and invasion capacities of MCF-7/ PTX cells and MDA-MB-453 cells. Consequently, as a novel next-generation taxane, SB-T-121205 appears to be a very promising lead compound for drug development.
Transgelin 2, located at chromosome 1q21-q25, is an important actin-binding protein responsible for the actin cytoskeleton dynamics (12). Abundant evidence has indicated that transgelin 2 exerts oncogenic activity. Transgelin 2 has been shown to be involved in lymph node metastasis, distant metastasis as well as tumor-lymph node-metastasis (TNM) staging system in colorectal cancer (CRC). Transgelin 2 may serve as a new biomarker for predicting progression and prognosis of CRC (14). Nohata et al (32) revealed that transgelin 2, directly regulated by miR-1, was downregulated by a siRNA and then decreased cell proliferation and invasion in human neck squamous cell carcinoma cells. In our models of paclitaxel-resistant breast cancer, we found that SB-T-121205 suppressed the transgelin 2 protein expression, which can explain the observed altered biological behavior of MCF-7/PTX cells. It has been generally accepted that the PI3K/Akt pathway participates in drug resistance, tumor migration, differentiation and apoptosis. Suppression of the PI3K/Akt pathway has been proven to be an efficient way to attenuate cell growth and migration (33,34). Wang et al (35) verified that the PI3K/Akt pathway was activated in cisplatinresistant lung cancer cells (A549/CDDP), and deactivation of Akt signaling pathway significantly suppressed Snail expression and subsequently induced a substantial decrease in migratory ability and invasiveness of A549/CDDP cells. Snail, a zinc-finger transcription factor, is known as a crucial regulator in the aggressive phenotype of EMT (36). It was supported that PI3K/Akt signal played an essential function during miR-519a-induced hepatocellular carcinoma cell proliferation and cell cycle progression (37). We found that the downregulation of transgelin 2 by SB-T-121205 caused repression of the PI3K/Akt pathway. Deactivation of Akt signal led to revitalization of the mitochondria apoptosis pathway and downregulation of Snail in MCF-7/PTX cells (Fig. 7), which appears to be an important contributor to the unique mechanism of action of SB-T-121205.
In conclusion, we found that SB-T-121205 enhances cell apoptosis, as well as inhibits the migration and invasion abilities of MCF-7/PTX cells, partly by targeting transgelin 2 and the PI3K/Akt pathway. We also found that SB-T-121205 downregulates the transgelin 2 expression. Deactivation of transgelin 2 can be further explored as a basis for new strategies for breast cancer treatment. These findings strongly indicate that SB-T-121205 is a highly promising lead compound for the development of next-generation chemotherapeutic agents in breast cancer treatment.