A Novel Small-Molecule YLT205 Induces Apoptosis in Human Colorectal Cells via Mitochondrial Apoptosis Pathway In Vitro and Inhibits Tumor Growth In Vivo

Colorectal cancer (CRC) continues to be one of the most common causes of cancer death both in men and women, being responsible for about 10% of total cancer-related mortality [1]. According to the report, there were over 102,480 new diagnoses and 50,830 deaths only in the United States in 2013 [2]. Chemotherapy, radiotherapy and surgery are the mainly strategies for colorectal cancer treatment at present, but not yet satisfactory for poor survival rates and liver metastases at the time of diagnosis [3]. Therefore, more effective strategy for CRC treatment is still an urgent need. Recently, the treatment of CRC is mainly focused on the deregulation of cell proliferation and apoptosis [4,5].

Apoptosis (programmed cell death) plays an important role in the balance between cell proliferation and cell death, and the deregulation of the apoptosis can lead to diseases, such as cancer [6]. Targeting apoptosis is a promising strategy in anticancer drug discovery [4,7], for cancer cells are highly dependent on aberration of the apoptosis signaling pathways to stay alive, and cancers are often resistant to chemotherapeutic agents that primarily work by inducing apoptosis [8,9]. Apoptosis signaling pathways can be divided into two pathways: cell death receptor-mediated extrinsic pathway and mitochondrial-mediated intrinsic pathway [10]. The intrinsic apoptosis pathway mainly act through mitochondria, and the important events include the loss of mitochondrial transmembrane potential, the release of cytochrome c, and the participation of pro- and anti-apoptotic Bcl-2 family proteins [11,12]. Intracellular death signals, such as DNA damage, toxins or depletion of ATP, can induce the release of cytochrome c from the inner membrane of mitochondria, which can subsequently interact with the Apaf-1 and ATP, which binds to pro-caspase-9, resulting in the cleavage of pro-caspase-9 and activation the effector caspase-3 and/or caspase-7 [13]. In the extrinsic pathway, replace with the death receptors by the ligands (e.g. TNFα and TNFR, FasL and Fas binding) leads to caspase-8 activation, and then cleavage and activation of downstream effector caspases, such as caspase-3 and caspase-7, resulting in chromatin condensation, DNA laddering and formation of apoptosis [14].

Our research group found a novel benzothiazole derivative, N-(5-bromopyridin-2-yl)-2-((6-(2-chloroacetamido)benzo[d]thiazol-2-yl)thio)acetamide (YLT205), which displayed anti-proliferative activity and induced apoptosis in vitro [15]. In this investigation, we demonstrate that YLT205 can induce apoptosis in colon cancer cells via the mitochondrial apoptotic pathway. By using xenograft models in athymic mouse, we determined that YLT205 also inhibited tumor growth in vivo by inducing apoptosis without causing toxic effects.

YLT205 was synthesized by our group (State Key Laboratory of Biotherapy, Sichuan University, Sichuan, China) [15], and its structural formula was shown in Fig. 1. The structure of YLT205 was determined by ¹H-NMR, ¹³C-NMR and ESI-MS analysis. The DMSO stocks of YLT205 was 20 mM and stored at -20 °C. For all in vitro assays, YLT205 was diluted to appropriate concentrations with the cell culture medium at final DMSO concentration of 0.1% (V/V). For in vivo experiment, YLT205 was dissolved in ultrapure water and Cremophor EL/ethanol (50:50, Sigma Cremophor EL, 100% ethyl alcohol).

3-(4,5-dimethyl-2-thiazolyl)-2,5-di-phenyl-2H-tetrazolium bromide (MTT), propidium iodide (PI), Rhodamine-123 (Rh123), Hoechst 33358, and dimethyl sulfoxide (DMSO) were purchased from Sigma Chemical Co (St. Louis, MO). Annexin V-FITC apoptosis detection kit was obtained from BD Biosciences (San Diego, CA). The antibodies against caspase-3 (17, 19, 35 kDa), caspase-9 (35, 37, 47 kDa), cytochrome c, Bcl-2 (26 kDa), Bax (20 kDa), Akt/p-Akt (Ser473, 60 kDa) and p44/42 MAPK/p-p44/42 MAPK (Tyr202/Tyr204, 44, 42 kDa) were purchased from Cell Signaling Technology Company (Beverly, MA). Antibody against β-actin was acquired from Santa Cruz Biotechnology Company (Santa Cruz, CA). The caspase inhibitor Z-VAD-FMK and PI3K/Akt inhibitor LY294002 were purchased from Beyotime (Beijing, China).

Human colon cancer cell lines HCT116, SW620, SW480, DLD-1, HCT-15, LS174T and other cells were purchased from American Type Culture Collection (ATCC, Manassas, VA), H1975 and SMMC-7721 cell lines were obtained from the China Center for Type Culture Collection (CTCCC, Wuhan, China). These cells were cultured in Dulbecco's modified Eagle medium (DMEM) or RPIM 1640 supplemented with 10% fetal bovine serum (Gibco, Auckland, N.Z), 100 units/mL penicillin, and 100 µg/mL streptomycin in a humid chamber at 37 °C under 5% CO₂ in atmosphere.

The MTT assay was used to measure the cell viability after YLT205 treatment. Briefly, cells were plated on 96-well microplates at a density of 2-5×10³ cells per well and cultured for 24 h. After various concentrations of YLT205 treatment for 48 h, 20 µL of MTT solution (5 mg/mL) was added to each well and incubation for 2-4 h at 37 °C. Then the medium was discarded and 150 µL of DMSO was added to each well to dissolve the formazan crystal produced by living cells. Ten minutes later, 96-well microplates were read on Spectra MAX M5 microplate spectrophotometer (Molecular Devices) at 570 nm for O.D. values [16].

For clonogenic survival assay, 400 to 1000 cells were seeded in a six-well plate, and treated with various concentrations of YLT205 (0∼0.625 µM) after 24 h, then the cells were re-fed once every 3 day for another 13 days with YLT205 and then fixed and stained with crystal violet.

Hoechst 33358 staining was used to detect morphological changes associated with apoptosis. After treatment with YLT205 for 24 h, cells were washed twice with PBS (Phosphate Buffered Saline, PH=7.4) and fixed with paraformaldehyde for 15 minutes. Then the cells were stained with 5 µg/mL Hoechst 33358 solutions. Morphological changes typical of apoptosis were observed under nuclear fluorescence microscopy (Zeiss, Axiovert 200, Germany).

To investigate the apoptosis-inducing effect of YLT205, we analyzed the percentage of apoptotic cells by FCM with propidium iodide (PI) staining or Annexin V-FITC/PI dual labeling. After treatment with YLT205 for 24 or 48 h as described above, harvested cells were washed twice with cold PBS and then stained with Annexin V-FITC and PI according to manufacturer's instructions, and then analyzed on a flow cytometer (Becton-Dickinson, USA) immediately. We also studied the apoptosis by PI single staining. Cells were harvested and fixed with 75% ice-cold ethanol, and then stained with hypotonic fluorochrome solution composed of PI at 50 µg/mL plus 0.1% Triton X-100 and then analyzed by FCM. Finally, the data was analyzed using Flow Jo software. Moreover, to investigate whether caspase and p44/42 MAPK are involved in YLT205-induced apoptosis in HCT116 cells, we treated cells with/without 2.5 µM YLT205 combined with 20 µM Z-VAD-FMK (caspase inhibitor) or 50 µM PD98059 (MAPK inhibitor), respectively. PI-stained was performed and analyzed by FCM. Finally, the data was analyzed using Flow Jo software.

The changes in mitochondrial transmembrane potential (ΔΨm) were analyzed by staining with Rh123 and detected by FCM as described previously [17]. Cell culture and drug treatment were done as described above. The harvested HCT116 cells were washed with cold PBS after incubation with Rh123 (5 µg/mL) at 37 °C for 30 min in the dark and then measured by flow cytometry.

Cells were treated with vehicle or YLT205 for 24 h. After being fixed with 4% paraformaldehyde for 15 min, the cells were incubated with blocking buffer (PBS containing 3% bovine serum albumin, 2% goat serum and 0.2% Triton X-100) for 1 h and incubated with antibodies against cytochrome c overnight. After washing twice with PBS, the cells were incubated with corresponding fluorescence-conjugated secondary antibodies for 1 h. Then changes in cytochrome c level were detected using nuclear fluorescence microscopy (Zeiss, Axiovert 200, Germany) [18].

To explore the mechanism of YLT205 on cell proliferation and cell apoptosis, we detected the change of protein using Western blot assay. Briefly, after treatment with YLT205 for 24 h, cells were washed twice with PBS after harvesting and centrifuged at 13,000 rpm for 15 min, followed by lysis in RIPA buffer for 30 min. The proteins were separated by SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The membranes were blocked for 1∼2 h at room temperature in 5% nonfat dry milk and incubated overnight at 4 °C with recommended dilute solution of primary antibodies, followed by incubation with horseradish peroxidase conjugated secondary antibody. Peroxidase-labeled bands were visualized using an ECL kit (Amersham, Piscataway, NJ). A monoclonal anti-β-actin antibody was used as a control.

All animal experiments were approved by the Institutional Animal Care and Treatment Committee of Sichuan University (Sichuan University, Chengdu, China). Female 5- to 6-week-old BALB/c athymic nude mice (Beijing HFK Bioscience Co. ltd., Beijing, China) were inoculated subcutaneously into the right flank regions with 1×10⁷ HCT116 cells or 1×10⁷ SW620 cells/mouse suspended in 100 µL of medium without serum and antibiotics. About a week later, mice bearing tumors around 100 mm³ were selected and then randomly divided into groups (8 mice per group). The mice were administrated in a dose schedule of YLT205 at 37.5, 75 and 150 mg/kg, or vehicle once a day by intraperitoneal injection. The mice were observed daily, and tumor size and body weight were measured every 3 days. Tumor volumes were calculated according to the formula: Tumor Volume = 0.5 × length × width². Growth inhibition was calculated from the start of treatment by comparison of the change in tumor volume for vehicle and treated group as before [19].

After treated with YLT205 for 14 days, tumors of HCT116 xenograft nude mice models were collected, fixed, processed, and embedded in paraffin for immunohistochemical analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. The tumor tissue was stained with H&E or treated with specific antibodies, like cleaved caspase-3. To explore whether YLT205 inhibited tumor growth by inducing apoptosis, apoptotic cells in tumor tissue was detected by TUNEL assay as described previously [20]. Three tumors per group were analyzed.

To investigate the potential side effects or toxicity of YLT205 on mice during the treatment, the etiological symptoms of the mice including mortality, body weight, diarrhea, anorexia and skin ulceration were monitored continuously [21]. Serological analysis, hematological analysis, and histological examination were carried out after dissection. Serological analysis and hematological analysis were detected by Hitachi 7200 Blood Chemistry Analyzer and a Nihon Kohden MEK-5216K Automatic Hematology Analyzer, respectively. Heart, liver, spleen, lung, and kidney of the mice were stained with hematoxylin and eosin (H&E).

Statistical analyses were carried out in Microsoft Excel or GraphPad Prism. P values were calculated by unpaired t test, and were considered significant if P < 0.05. Data are displayed as means±SD/SEM from three independent experiments in the figures.

To test the effect of YLT205 on cancer cell viability, a panel of cancer cell lines of different histological types was treated with YLT205 for 48 h, and the IC₅₀ values were expressed. As shown in Table 1, IC₅₀ values ranged from 0.59 to 13.18 μM, and a comparison among the examined 23 cell lines showed that human rectal cancer cell lines were most sensitive to YLT205. Thus, we chose human colorectal cancer cells to further study for the YLT205 exerted antitumor mechanism. Furthermore, exposure of HCT116, SW620, SW480, DLD-1, Ls174T and HCT-15 cells to YLT205 for 12 h, 24 h and 48 h, respectively, resulted in decrease of the cell proliferation (Fig. 2A). These data suggested that YLT205 inhibited colorectal cancer cells proliferation in a concentration- and time-dependent manner.

To further determine the effect of YLT205 on cell growth, we conducted clonogenic assay after YLT205 treatment. Fig. 2B and Fig. 2C show that colony formation of HCT116 and SW620 cell lines were inhibited in a concentration-dependent manner after exposure to YLT205. When the concentration reached to 0.625 µM, nearly no colony formation was found. These results were consistent with the MTT data and further suggested that YLT205 could inhibit cell growth of colorectal cancer cell lines.

To examine the possible mechanism of YLT205 to inhibit cell viability, apoptosis was examined after YLT205 treatment of colorectal cancer cells. To such regard, we applied flow cytometric analysis and Hoechst 33358 nuclear staining. As shown in Fig. 3B, the percentage of sub-G1 cells in HCT116 cells increased from 2.13% to 4.42%, 21.30%, 49.55% and 83.70% after 0.3125 µM, 0.625 µM, 1.25 µM, 2.5 µM YLT205 treatment for 24 h, respectively, indicating that YLT205 was able to induce apoptosis in a concentration-dependent manner. Similarly, in SW620 cells, the percentage of apoptotic cells was increased from 3.60% to 5.13%, 19.03%, 25.36% and 44.88% after treatment various concentration YLT205 for 48 h (Fig. 3B), respectively. Furthermore, as shown in Fig. 3A, the features of apoptotic cells were shown after YLT205 treatment in HCT116 and SW620 cells. For instance, brilliant-blue fluorescence stained and condensed nuclei, cytoskeletal collapse, reduction of cell count and nuclear fragmentation were evident.

To further confirm the apoptosis in the cell death result, we applied another method by Annexin V-FITC combined with PI fluorescence staining, which was determined by flow cytometry. As shown in Fig. 4, YLT205 dose-dependently increased the percentage of apoptotic cells (positive Annexin V-FITC and negative PI staining) after exposure to various concentrations of YLT205 for 24 h in HCT116 cells and 48 h in SW620 cells as well.

Rh123, a fluorescence dye, was used to detect the ΔΨm change of cells after YLT205 treatment, and cytochrome c release was examined through immunofluorscence. As shown in Fig. 5B, YLT205 treatment for 24 h led the loss of ΔΨm potential in HCT116 cells in a dose dependent manner, showing a decreased ΔΨm from 95.29% in the vehicle to 58.64% in the 2.5 µM treatment group. The loss of mitochondrial membrane potential would be the release of cytochrome c from the mitochondria to the cytosol. The result of the immunofluorscence detection of cytochrome c showed that the level of cytochrome c significantly increased in the cytosols of HCT116 cells after treatment with 2.5 µM YLT205 (Fig. 5C), which was in line with the ΔΨm change.

To further conform the characterization of YLT205-induced apoptosis, some apoptosis-related proteins were detected by Western blot, including caspase family proteins, Bcl-2 family proteins, Akt and MAPK. Firstly, we examined changes in the proteolytic processing of caspase-3 and caspase-9. As shown in Fig. 5A, expression of pro-caspase-3 and pro-caspase-9 in HCT116 cells was decreased significantly and the levels of their cleaved forms were increased after YLT205 exposure for 24 h. Next, we examined Bcl-2 family proteins, including Bcl-2 and Bax. Fig. 5D showed that the expression of Bcl-2 was decreased significantly while that of Bax was increased after YLT205 treatment.

In addition, the expression levels of p44/42 MAPK and Akt, which play important roles in cell proliferation and apoptosis, were examined. As shown in Fig. 5E, YLT205 treatment suppressed the expression of phosphorylated p44/42 MAPK without affecting its total expression level, but there was no clearly change in the level of phosphorylated Akt.

To explore which type of apoptotic pathway is a predominant role in YLT205-induced apoptosis, Z-VAD-FMK (caspase inhibitor) and PD98059 (MER/ERK inhibitor) were used. As shown in Fig. 5F, compared with YLT205 treatment alone, treatment with 2.5 µM YLT205 combined with 20 µM Z-VAD-FMK decreased the percentage of apoptotic cells from 80.38% to 20.58% while combination with 50 µM PD98059 had only a slight effect on apoptosis induced by YLT205 treatment.

To study the antitumor activity of YLT205 in vivo, HCT116 or SW620 tumor bearing BALB/c nude mice were established and treated with YLT205 at doses of 37.5, 75 and 150 mg/kg/day, respectively. As shown in Fig. 6A, compared with the vehicle group, YLT205 substantially suppressed tumor growth in a dose-dependent manner, with the inhibition rate of tumor volumes at 68.8% (SW620) and 51.3% (HCT116) at the dose of 150 mg/kg. Moreover, YLT205 treatment was well tolerated and body weight of mice was change slightly (Fig. 6B), and no other adverse effects, such as toxic death and skin ulcerations, were observed in YLT205 groups.

To elucidate the mechanism of YLT205 to inhibit tumor growth in vivo, immunohistochemical analysis and TUNEL staining were done. As shown in Fig. 7A, hematoxylin-eosin staining (H&E) revealed the regression of tumors in YLT205 treated animals. Besides, YLT205 remarkably decreased the number of proliferating cells in tumor tissues as indicated by cell cycle marker Ki67 and increased the number of cleaved caspase-3-positive cells in tumors compared with vehicle group. Moreover, TUNEL-positive cells in YLT205-treated group were higher than that in vehicle group with the percentage rise from 2% to 33% (Fig. 7B and Fig. 7C), Showing that YLT205 suppress tumor growth in vivo through inducing cell apoptosis.

To further investigate the safety profile of YLT205, all the mice in SW620 models sacrificed after 28 days treatment followed by toxicity evaluations. Serum biochemistry and hematological of the mice did not show any pathological changes (Table 2). Moreover, microscopic examination of heart, liver, spleen, lung and kidney revealed that there were no toxic pathological changes after YLT205 treatment (Fig. 7D).

YLT205 is a novel chemical structure which differs from the apoptosis inhibitors currently in clinical research [15]. This study firstly demonstrated that YLT205 can induce apoptosis in human colorectal adenocarcinoma cells via the mitochondrial apoptotic pathway in vitro and inhibited tumor growth in vivo.

There are two classic apoptotic pathways, including the extracellular pathway and the intracellular pathway. In both pathways, caspase-3 plays a central role [22] and can be activated by upstream effector proteins, such as caspase-9, which could arouse the activation of mitochondrial intrinsic pathway [23]. The cleavage of caspase-9 and caspase-3 were observed after YLT205 treatment, and these results are consistent with our initial hypothesis that YLT205 induces apoptosis via the mitochondrial intrinsic pathway. In order to further investigate whether YLT205-induced apoptosis is caspase-dependent or not, the irreversible inhibitor, Z-VAD-FMK (caspase inhibitor) was used. We observed that Z-VAD-FMK significantly decreased the apoptosis cells of the YLT205-induced in HCT116 cells, suggesting that caspase plays a role in YLT205-induced apoptosis.

In the intrinsic apoptotic pathway, many important events are closely related with the mitochondria, including the disruption of the mitochondrial transmembrane potential and the induction release of cytochrome c [10,11]. In general, cytochrome c is localized in the mitochondrial membrane and would be released into the cytosol when intrinsic apoptosis was induced [11]. Then it would lead to cleavage of caspase-9, which would finally activate downstream caspase-3 to initiate apoptosis [24]. In this study, we observed substantial reduction in ΔΨm by FCM and an increase in the release of cytochrome c from mitochondria into the cytosol after YLT205 treatment.

Bcl-2 family is the center regulator in mitochondrial apoptosis pathway, and this family includes the anti-apoptotic Bcl-2 and pro-apoptotic Bax [12], increasing in pro-apoptotic protein but decreasing in anti-apoptotic protein would stimulate the release of cytochrome c [25]. In our study, anti-apoptotic protein Bcl-2 was down-regulated while pro-apoptotic protein Bax was up-regulated in HCT116 cells after treated with YLT205. We assume that the change of the Bcl-2 family proteins is involved in the release of cytochrome c and may provide a selective growth advantage and confer resistance to YLT205.

In addition, other signaling proteins, such as phosphatidylinositol 3-kinase (PI3K)/Akt and MAPK pathways, are involved in apoptosis pathways [26]. Both Akt and MAPK pathways play important roles in promoting cell proliferation and modulating cancer prevention and treatment through apoptosis. Our results showed that phosphorylation of p44/42 MAPK was decreased after YLT205 treatment while phosphorylation of Akt was not affected. But, we observed that the percentage of apoptotic cells only had a slight effect on YLT205-induced apoptosis after adding the MER/ERK inhibitor PD98059. Combined this results and the significant decrease in apoptosis cells after Z-VAD-FMK addition, we conclude that caspases play an important role in YLT205-induced apoptosis.

Furthermore, tumor growth inhibition in vivo was evaluated in tumor-bearing nude mice. The dose of 150 mg/kg/day treatment by YLT205 was able to inhibit tumor growth at the inhibitory rate of 68.8% and 51.3% in human colon cancer SW620 and HCT116 models, respectively. Besides, the results of immunohistochemistry and TUNEL assay revealed that YLT205 could induce apoptosis in the tumor sections, as shown by the TUNEL-positive and cleaved caspase-3-positive cells. YLT205 also showed inhibition of cell proliferation demonstrated by a decrease in Ki67-positive cells. Furthermore, YLT205 was well tolerated in mice in the study. In conclusion, YLT205 not only inhibited cancer cell proliferation in vitro, but also had an effective inhibition of tumor growth in vivo without causing obvious toxicities.

Taken together, we demonstrated, for the first time, that YLT205 could inhibit human colon cancer growth both in vitro and in vivo, and induce human colon cancer cells apoptosis via mitochondrial intrinsic pathway. The mechanism of apoptosis was related to down regulation of Bcl-2 and conversely up regulation of Bax, along with the disruption of mitochondrial membrane, followed by release of cytochrome c from mitochondria to cytosol, which eventually leads to cleavage of executor caspase-3 and the apoptosis. YLT205 also showed antitumor activities in the colon cancer xenograft in vivo with no obvious toxicity, suggesting that it may be a relatively nontoxic adjuvant treatment drug for cancer therapy while its anti-tumor activities in other cancer types and the more precise mechanisms are still needed in future study.