The preclinical analysis of TW-37 as a potential anti-colorectal cancer cell agent

TW-37 is a novel, potent and non-peptide Bcl-2 small-molecule inhibitor. Its activity in colorectal cancer (CRC) cells is studied. In both HCT-116 cells and primary human colon cancer cells, treatment with TW-37 at only nM concentration efficiently inhibited cell survival and proliferation. TW-37 also induced caspase-3/9 and apoptosis activation in CRC cells. Feedback autophagy activation was observed in TW-37-treated CRC cells. Reversely pharmacological autophagy inhibition or Beclin-1 knockdown by targeted-shRNA potentiated TW-37-induced apoptosis and killing of CRC cells. In vivo, intravenous injection of TW-37 inhibited HCT-116 tumor growth in mice. TW-37’s anti-tumor activity was further potentiated against Beclin-1-silenced HCT-116 tumors. Together, targeting Bcl-2 family protein by TW-37 efficiently inhibits CRC cell growth in vitro and in vivo. Inhibition of feedback autophagy activation could further sensitize TW-37.


Introduction
Colorectal cancer (CRC) is the third most-common human malignancy [1,2,3,4]. Each year, it is estimated that over one and half million new cases of CRC will be diagnosed [1,2,3,4]. The current CRC treatment options include the combinations of surgery resection, chemo-therapy and/or radio-therapy [5,6]. However, for the patients with advanced, recurrent or metastatic CRC, the prognosis is often poor [5,6].

Chemicals and reagents
TW-37 was provided by Selleck LLC (Nanjing, China). Three-methyladenine (3-MA) and chloroquine (Cq) were obtained from Sigma (Shanghai, China). All the antibodies in this study were purchased from Cell Signaling Tech (Shanghai, China). Puromycin was also purchased from Sigma (Shanghai, China). The medium an reagents for cell culture were provided by Gibco (Shanghai, China).

Primary culture of human cells
A total of two colon cancer patients (Both female, 56/61 years old, undergoing whole colon cancer resection), administrated at Southwest Hospital, the Third Military Medical University, were enrolled in this study. The fresh colon cancer tissues and surrounding normal epithelial tissues were separated very carefully under the microscopy. Tissues were then minced, washed, and digested by collagenase I (Sigma). Digestions 2-5 were neutralized, pooled, and filtered. Single cell suspensions of primary human colon cancer cells and colon epithelial cells were cultured in the described medium for primary human cells [14,15]. Two primary human colon cancer cell lines and one primary colon epithelial cell line were established in this study. The protocols using human tissues were conducted according to the principles of Declaration of Helsinki, and were approved by the Ethics Review Board (ERB) of The Third Military Medical University. Written-informed consent was obtained each participant.

MTT assay
The routine MTT (Sigma) dye assay was performed to test cell viability. MTT optical density (OD) value at 590 nm was recorded.

Colony formation assay
Following the TW-37 treatment, HCT-116 cells were re-suspended in complete medium with 0.5% of agar (Sigma), which were then added on the top of a pre-solidified six-well plate. TW-37-containing medium was renewed every two days. After 8 days incubation, the number of surviving colonies was counted.

BrdU incorporation assay of cell proliferation
HCT-116 cells were initially plated into 96-well plate (1 ×10 4 cells/well). After the indicated TW-37 treatment, HCT-116 cells were further incubated with BrdU (10 μM, Cell Signaling Tech, Shanghai, China) for additional 12 hours. Cells were then fixed, and BrdU incorporation was determined via a commercial available enzyme-linked immunosorbent assay (ELISA) kit (Cell Signaling Tech). BrdU ELISA OD at 405 nm was recorded.

TUNEL staining assay of cell apoptosis
Cells with the indicated TW-37 treatment were fixed by 4% cold formaldehyde (Sigma),followed by incubation with dUTP nick end-labeling (TUNEL) fluorescence dye (Roche, Indianapolis, IN). Cells were then subjected to observation under a confocal microscope (Leica TCS SMD FCS). TUNEL ratio was recorded. For each condition, at least 100 nuclei in five random microscope views (1:100) were analyzed.

Single-stranded DNA (ssDNA) ELISA assay of apoptosis
The production of ssDNA is known as a key marker of cell apoptosis [17]. After TW-37 treatment, cellular ssDNA content was analyzed via the ELISA format using the commercial available ssDNA kit [18]. The detailed protocol was described previously [18]. The ssDNA ELISA OD at 450 nm was recorded to quantify cell apoptosis.

Western blotting assay
Following treatment, cells were incubated with RIPA lysis buffer (Biyuntian, Wuxi, China). Equal amount of lysate proteins (40 μg per condition) were separated by the SDS-PAGE gels, and were transferred to the polyvinylidenedifluoride (PVDF) membrane (Millipore, Shanghai, China). After blocking, the blot was incubated with designated primary and secondary antibodies. The enhanced chemiluminescence (ECL) reagents (Pierce, Shanghai, China) were added to the blots, and signals were developed under the hyper-X-Ray films. Total gray of each band was quantified using the ImageJ software (NIH), it was always normalized to loading control, β-Tubulin.

Beclin-1 shRNA
Two distinct lentiviral Beclin-1 shRNAs (a/b) as well as the scramble control shRNA were gifts from Dr. Zhu [19].The detailed protocol of lentiviral shRNA infection was described early [19]. After infection, puromycin (5.0 μg/mL) were added to select stable cells for 4-6 days. Knockdown of Beclin-1 in the stable cells was verified by Western blotting assay.

Tumor xenograft assay
The female severe combined immuno-deficient (SCID) nu/nu mice (4-5 weeks old, 17-18 grams in weight) were purchased from the Animal Center of The Third Military Medical University. Animals were housed in temperature-and humidity-controlled cages, with free access to water and rodent food on a 12-h light/dark cycle. Mice were injected subcutaneously (s.c.) with HCT-116 cells (5×10 6 cells per mouse). When the tumors reached the volume of around 100 mm 3 , the mice were divided into four groups. Mice were treated via intravenous injection (i.v.) with TW-37 (10 mg/kg body weight, daily for 15 days) or saline vehicle control. TW-37 was always freshly prepared before injection. Estimated tumor volume was calculated by: π/ 6 × larger diameter × (smaller diameter) 2 . All animal studies were performed in accordance with the standards of ethical treatment approved by the Institutional Animal Care and Use Committee (IACUC) and ERB of The Third Military Medical University. Animals were observed on daily bases. Humane endpoints include a loss of over 15% of body mass, a tumor greater than 1.3 cm, severe fever, vomiting or skin problems or inability to ambulate or rise for food and water. If reaching these endpoints, CO 2 inhalation was used for euthanasia of animals. All surgical procedures were performed under anesthesia by intramuscular injection of 50% ketamine, 38% xylazine, and 12% acepromazine maleate (0.02 mL). All efforts were made to alleviate suffering.

Statistics analysis
Data were expressed as mean ± standard deviation (SD). A p value, calculated by ANOVA (SPSS16.0), of less than 0.05 was considered statistically significant. The concentrations of reagents applied and the treatment durations were chosen based on published literatures and results from our pre-experiments. IC-50 was calculated also by SPSS software.

TW-37 inhibits CRC cell survival and proliferation
HCT-116 CRC cells were treated with TW-37 at 10-1000 nM. MTT assay was performed to test cell viability. Results in Fig 1A demonstrated that TW-37 efficiently inhibited HCT-116 cell survival. The novel Bcl-2 inhibitor displayed both time-and dose-dependent response in decreasing HCT-116 cell survival (Fig 1A). Treatment with TW-37 for 72 hours was more dramatic than 48 hours in suppressing HCT-116 cells (Fig 1A). The IC-50, or the concentration that led to 50% cell viability reduction, was close to 1000 nM at 48 hours, and 100-300 nM at 72 hours ( Fig 1A). The colony formation assay results in Fig 1B showed that TW-37, at 30-1000 nM (treatment renewed every two days), also efficiently decreased the number of viable HCT-116 colonies. The effect of TW-37 was again dose-dependent ( Fig 1B).
To study the potential effect of TW-37 on HCT-116 cell proliferation, BrdU incorporation ELISA assay was applied. Quantified results in Fig 1C demonstrated that BrdU ELISA OD was significantly decreased in TW-37 (30-1000 nM)-treated HCT-116 cells, indicating proliferation inhibition. The TW-37's anti-proliferative activity was again dose-dependent ( Fig 1C). Notably, to exclude the possible influence of cytotoxicity, cells were only incubated with TW-37 for 24 hours for analyzing BrdU incorporation (Fig 1C).
Two lines of primary human colon cancer cells were also established. Both were derived from patient cancer tissues and were named as "Colon can-1/2". MTT assay results in Fig 1D confirmed that treatment with TW-37 (300 nM, 72 hours) was cytotoxic to both lines of primary colon cancer cells. Intriguingly, the very same TW-37 treatment was safe and non-cytotoxic to the primary colon epithelial cells (non-cancerous cells, Fig 1D). These results suggest a selective response of TW-37 in cancerous cells.

TW-37 provokes apoptosis in CRC cells
In order to study the effect of TW-37 on CRC cell apoptosis, the caspase-3/-9 activities were examined. As displayed in Fig 2A, treatment with TW-37 at 30-1000 nM significantly increased the activities of both caspase-3 and caspase-9 in HCT-116 cells. Furthermore, in TW-37-treated HCT-116 cells, cleavages of caspase-9 and poly (ADP-ribose) polymerase (PARP) were observed (Fig 2B), which were followed by the formation of single strand DNA (ssDNA) (Fig 2C). Moreover, TUNEL assay results in Fig 2D showed that the percentage of cells with TUNEL-positive nuclei was dramatically increased following TW-37 (30-1000 nM) treatment. These results confirmed the activation of apoptosis in TW-37-treated HCT-116 cells.
In the two lines of primary colon cancer cells, treatment with TW-37 (300 nM, 48 hours) again induced significant apoptosis activation (TUNEL assay, Fig 2E). TW-37 was however non-apoptotic when added to the colon epithelial cells (TUNEL assay, Fig 2E). Collectively, our results indicate thatTW-37 provokes apoptosis in CRC cells.
To study the potential function of autophagy in TW-37-induced activity against HCT-116 cells, pharmacological strategy was first applied. Two well-established autophagy inhibitors, including 3-methyladenine (3-MA) [26] or chloroquine [27], were applied. As displayed in  (Fig 3C and 3D). Notably, treatment with 3-MA or Cq alone only induced minor viability reduction and apoptosis in HCT-116 cells.
In the primary colon cancer cells ("Colon can-1"), treatment of TW-37 (300 nM, 24 hours) also induced above change in the expressions of the autophagy proteins ( Fig 3E). More importantly, TW-37-induced viability reduction ( Fig 3F) and apoptosis ( Fig 3G) were again potentiated with co-treatment of 3-MA, the autophagy inhibitor. Collectively, these results suggest that TW-37 activates feedback autophagy in CRC cells, and pharmacological inhibition of autophagy significantly sensitizes TW-37-induced killing of CRC cells.

Fig 2. TW-37 provokes apoptosis in CRC cells. HCT-116 CRC cells (A-D)
, the primary human colon cancer cells (two lines, "Colon can-1/ 2", E) and the primary human colon epithelial cells ("Colon Epi1", E) were either left untreated ("C") or stimulated with TW-37 at applied concentrations (10-1000 nM), cells were further cultured in TW-37-containing-medium for designated time; Cell apoptosis was tested by the listed assays mentioned in the text. Cleaved-Caspase-9 and Cleaved-PARP were quantified and normalized to β-Tubulin (B). Data were presented as mean (n = 5) ± standard deviation (SD). *p<0.05 vs. "C". Experiments in this figure were repeated three times, and similar results were obtained.  control mice) to 13.89 ± 3.23 mm 3 per day after TW-37 injection (Fig 5B). The weight of the tumors (At Day-35) in TW-37-treated mice was also lighter than that of the control mice ( Fig 5C). Thus, TW-37 i.v. injection inhibited HCT-116 tumor growth in vivo.

Discussion and conclusion
The anti-apoptosis Bcl-2 family proteins, including Bcl-2, Mcl-1, Bcl-xL, are often over-expressed in CRC [29,30] and many other malignancies [8,9]. They are important for CRC progression and chemo-/ratio-resistance [8,9]. TW-37 is a newly-developed small-molecule Bcl-2 inhibitor, it targets multiple members of anti-apoptosis Bcl-2 proteins [8,9]. In the current study, treatment with TW-37 at only nM concentration inhibited survival and proliferation of both HCT-116 cells and primary human colon cancer cells. Meanwhile, TW-37 induced activation of caspase-3/9 and apoptosis in the CRC cells. In vivo, intravenous injection of TW-37 inhibited HCT-116 tumor growth in mice. Our results suggest that targeting Bcl-2 family protein by TW-37 could be a fine strategy to inhibit CRC cells.
Autophagy begins with the formation of the double membrane vesicle structures in the cytoplasm, named autophagosomes [31,32], those are responsible for degrading organelles and other cytoplasmic contents via acidic lysosomal hydrolases [31,32]. The process will provide , scramble non-sense control shRNA ("shC"), as well as the parental control cells ("Par"), were treated with/out TW-37 (300 nM) for applied time; Expression of listed proteins was shown (A, data were quantified), Cell survival (MTT assay, B) and apoptosis (ssDNA ELISA assay, C) were also tested. Data were presented as mean (n = 5) ± standard deviation (SD). *p<0.05 vs. "C". # p<0.05 vs. "shC" cells. Experiments in this figure were repeated four times, and similar results were obtained. nutrition and energies for cell to survive [31,32]. Dysregulation of autophagy has been detected in different types of cancers, which is associated with cancer cell progression and chemo-resistance [32,33]. Meanwhile, multiple cancer-killing agents of different mechanisms of action were demonstrated to induce feedback and cytoprotective autophagy activation [32,33]. Reversely, autophagy inhibition via genetic or pharmacologic methods, could significantly sensitize the anti-cancer activity by the agents [20,21].