A Novel Imidazopyridine Derivative Exerts Anticancer Activity by Inducing Mitochondrial Pathway-Mediated Apoptosis

Background Cancer remains a major clinical challenge because of the lack of effective drug for its treatment. To find out novel cancer chemotherapeutic molecules, we explored the anticancer effect of novel imidazopyridine compound 9i and also investigated the underlying molecular mechanism. Methods Human cervical cancer cell (HeLa) viability was measured by an MTT assay after treatment with compound 9i. Clonogenicity of HeLa cells was investigated by an in vitro colony formation assay. Cell death was visualized by propidium iodide (PI) staining. Fluorescence-activated cell sorting (FACS) was used to determine apoptosis and mitochondrial membrane potential in HeLa cells. The expression level of apoptosis-related proteins was also determined by western blot. Results Compound 9i suppressed HeLa cell viability in a time- and dose-dependent manner. Compound 9i induced mitochondrial outer membrane permeabilization (MOMP), activated caspase cascade, and finally resulted in apoptosis. Conclusion Compound 9i induces mitochondrial pathway-mediated apoptosis in human cervical cancer cells, suggesting that 9i could be a potential lead compound to be developed as a cancer therapeutic molecule.

Apoptosis is programed cell death which is divided into two major subtypes, extrinsic and intrinsic apoptosis. Intrinsic apoptosis is initiated by mitochondrial outer membrane permeabilization (MOMP) [7]. Mitochondria are cellular energy generators, which are vital organelles in all kinds of eukaryotic cells [8]. At normal condition, mitochondria generate ATP to supply energy for intracellular metabolic pathways [9]. Upon a cytotoxic stimulus, mitochondrial outer membrane integrity was damaged (also known as MOMP) which results in cytochrome c release into the cytosol. The release of cytochrome c activates caspase-9 which activates effector caspases (caspase-3, caspase-6, and caspase-7), finally resulting in apoptosis [10]. MOMP is enhanced by proapoptotic proteins Bcl-2-associated X (BAX) and Bcl-2 antagonist killer (BAK); meanwhile, it is suppressed by antiapoptotic Bcl-2 family proteins (Bcl-2, Bcl-XL) [11,12].
Here in this study, we further explored the molecular mechanism of compound 9i inducing apoptosis in the human cervical cancer cell line HeLa. We found that compound 9i decreased HeLa cell viability by inducing apoptosis. The following experiments revealed that 9i upregulated proapoptotic proteins, induced MOMP, activated caspase cascade, and finally resulted in apoptosis. Taken together, the current research demonstrates that novel imidazopyridine compound 9i induces mitochondrial pathway-mediated apoptosis in HeLa cells, which is the major incentive for its anticancer effect on cervical cancer cells. It suggests the potential role of 9i as a promising novel anticancer agent.

Materials and Methods
2.1. Chemicals. Small molecular compound 9i was synthesized by the chemical synthesis lab of IATTI as previously described [6]. Other regular chemical reagents were purchased from Sigma-Aldrich.

Cell
Culture and Compound Treatment. The human cervical cancer cell line HeLa and noncancerous cell lines HEK293T and PNT1A used in this study were obtained from Cobier Biotechnology (Cobier, Nanjing, China). HeLa cells and PNT1A cells were maintained in RPMI 1640 medium (HyClone, SH30809.01, USA), HEK293T cells were maintained in DMEM (HyClone, SH30022.01, USA), with 10% fetal bovine serum (FBS, Gibco, 10100147, Australia) and 1% penicillin-streptomycin (Gibco, 15140122, Australia) added, and the cells were kept in a humidified atmosphere of 5% CO 2 at 37°C. Compound 9i was synthesized in our lab as previously described. Stock solution of compound 9i was prepared in dimethyl sulfoxide (DMSO) and diluted in culture medium whenever needed. The final concentration of DMSO was less than 0.5% which did not affect cell survival.
2.3. Cell Viability Assay. The HeLa cells were cultured in 96well plates with 3 × 10 3 cells seeded. After 16-hour incubation, the medium was removed and 200 μL fresh medium containing the indicated concentrations of compound was added. The compound-treated cells were cultured for another 24 hours, 48 hours, and 72 hours as indicated in the figure. The viability of cells was measured by an MTT assay based on the previous description [13]. 20 μL of MTT solution (5 mg/mL in PBS) was added to each well and then incubated for another 4 hours. The medium was removed, and 150 μL of DMSO was added to dissolve the formazan crystals. The optical density (OD) was measured with a microplate reader (BioTek, Winooski, VT, USA) at an absorbance wavelength of 570 nm. The survival rate of the compound-treated cells was compared to the equal amount of DMSO-treated cells. The IC 50 of compound 9i was measured after 48 hours of compound treatment.

Colony Formation
Assay. The colony formation assay was based on the previous description [14]. The cervical cancer cell HeLa was seeded onto 6-well plates at a density of 500 cells per well containing 2 mL medium. After 16-hour incubation, cells were treated with compound 9i at concentrations of 0, 5, 10, and 20 μM for 48 hours. Cells were cultured in complete medium without 9i for another 14 days, and cell culture medium was changed with fresh medium every 2 days. The cells were washed twice with PBS and fixed with 4% paraformaldehyde for 30 minutes, followed by staining with 0.5% crystal violet (Beyotime, C0121, Shanghai, China). Colonies were documented with an Epson scanner.
2.5. PI Staining Assay. HeLa cells (1 × 10 5 cells/well) were seeded into 6-well plates. After 16 hours of attachment, the cells were treated with 5 μM compound 9i or DMSO for 24 hours. The cells were washed twice with PBS, followed by staining with PI (0.1 μg/mL) and DAPI (0.5 μg/mL) for 15 minutes at room temperature in the dark [15]. The images were obtained using an inverted fluorescence microscope (Olympus, Tokyo, Japan).
2.6. Apoptosis Assay. Cell apoptosis assays were based on the previous description [16]. Flow cytometry was performed with an annexin V-FITC apoptosis detection kit (Beyotime, C1063, Shanghai, China) according to the manufacturer's protocol. Cells were stained with propidium iodide (PI)/annexin V-FITC (annexin V-fluorescein isothiocyanate) and analyzed by flow cytometry. HeLa cells were treated with different concentrations of 9i for 48 hours. All cells were harvested and washed twice with cold PBS, followed by incubation with PI and annexin V-FITC for 15 minutes at room temperature in the dark. The cell apoptosis was assessed by flow cytometry (Becton Dickinson, Accuri™ C6, USA).

Mitochondrial
Membrane Potential (ΔΨm) Assay. The mitochondrial membrane potential was detected with rhodamine 123 (Sigma-Aldrich, USA) which is a ΔΨm-specific fluorescent probe. The experiments were performed as previously described [17]. Briefly, HeLa cells were treated with compound 9i for 24 hours at different concentrations (0, 5, 10, and 20 μM). The cells were washed twice with PBS, followed by incubation with rhodamine 123 and PI for 30 minutes at 37°C in the dark. The fluorescence intensities were analyzed using flow cytometry (Becton Dickinson, Accuri™ C6, USA) with the setting of FL1A at 530 nm and FL2H at 585 nm.
2.9. Statistics. All data were presented as mean ± SD from three independent experiments. All statistical analyses were carried out using a GraphPad Prism 5 software program for Windows (GraphPad Software, Inc.). Statistical significance was determined by Student's t-test ( * p < 0:05, * * p < 0:01).

Compound 9i Inhibits Cervical Cancer Cell Viability in a
Time-and Concentration-Dependent Manner. Our previous study found that a series of novel imidazopyridine analogues have anticancer activity in various tumor cell lines [6]. We want to further explore the molecular mechanism of the anticancer effect of these compounds. Compound 9i (Figure 1(a)) was chosen for further research as it has the best anticancer effect.
To evaluate the anticancer effect of compound 9i, cell viability was measured after compound 9i treatment in human cervical cancer HeLa cell lines. The half maximal inhibitory concentration (IC 50 ) of compound 9i after 48-hour intervals was 10.62 μM in HeLa cells (Figure 1(b)). To explore the Survival rate equals to compound 9i-treated cells compared to vehicle control cells; DMSO was used as a vehicle control. Data is represented as mean ± SD (n = 3). Significance was tested by Student's t-test ( * p < 0:05, * * p < 0:01).
dosage and temporal effect of compound 9i on cancer cell survival, HeLa cells were treated with compound 9i at different concentrations (0, 2.5, 5, 10, 20, 40, and 80 μM) for 24, 48, and 72 hours, respectively, and the cell survival rate was measured. Cell viability decreases as the concentration of compound 9i increased after 24-hour, 48-hour, and 72hour intervals, respectively. The cell viability also decreases as the treated time increases under each concentration of compound 9i treatment (Figure 1(c)). Compound 9i did not affect normal human cell viability in human embryonic kidney 293T (HEK293T) cells and normal prostate epithelium PNT1A cells ( Figure S1). The results indicate that 9i decreases the HeLa cell survival rate in a time-and concentration-dependent manner.

Compound 9i
Inhibits Colony Growth. To explore the effect of compound 9i on clonogenicity, we conducted an in vitro colony formation assay. HeLa cells were treated with different concentrations of compound 9i for 48 hours and cultured in fresh medium for another 14 days to let the colony grow. Colony size and numbers were dramatically decreased as the concentration of compound 9i increased ( Figure 2). The results indicate that compound 9i inhibits human cervical cancer colony growth.

Compound 9i
Induces Cell Death. The above results indicated that compound 9i inhibited tumor cell viability; we want to know whether 9i induces cell death. Propidium iodide (PI) is a small fluorescent molecule that binds to DNA. PI cannot pass through the cell membrane when the plasma membrane is intact. When the cell is undergoing cell death, the cell membrane becomes permeable which makes it possible for PI to traverse into the nucleus. So the dead cell can be stained and marked by PI. HeLa cells were treated with compound 9i for 24 hours and stained with PI, compared with control (Figure 3(a)), compound 9i treatment slightly decreased the cell number (Figure 3(d)). Part of the cells engulfed PI and emitted red fluorescence (Figure 3(e)), while the vehicle control cells were not stained with PI ( Figure 3(b)). The results indicated that 9i induces cell death in HeLa cells.

Compound 9i
Induces Apoptosis in a Concentration-Dependent Manner. We noted that in the dead cell stained

BioMed Research International
with PI, the chromatin condensed to form a bright blue particle (Figures 3(d) and 3(f)), which is a remarkable process of apoptosis. The vehicle-treated HeLa cells showed no condensation or PI staining (Figures 3(a) and 3(c)). To validate whether compound 9i induces apoptosis, we conducted fluorescence-activated cell sorting (FACS) to measure the apoptotic cells after annexin V-FITC/PI double staining. We found that as the concentration of compound 9i increased, the apoptosis rate of the HeLa cells increased (Figures 4(a) and 4(b)).
Apoptosis is programed cell death regulated by cysteine aspartate protease (caspase) family proteins. We measured the protein levels of caspases and their activation forms in compound 9i-treated HeLa cells by western blot. The protein level of full-length caspase-3 decreased, while its activation from cleaved caspase-3 significantly increased after 9i treatment (Figure 4(c)), implying that caspase-3 is cleaved and activated by initiator caspase-9. Consistently, we found that the total level of caspase-9 decreased and cleaved caspase-9 increased as the concentration of 9i increased (Figure 4(c)).      3). Significance was tested by Student's t-test ( * * p < 0:01, * * * p < 0:01 versus DMSO-treated cells). (c) Western blot was used to determine the expression level of apoptosis-related proteins in DMSO-or compound 9i-treated HeLa cells. The intensity of the bands was measured, and the fold change of the intensity was compared with that of the control and indicated below the bands. The expression of Bcl-xL was statistically analyzed in Figure S2b. Tubulin was used as a loading control. The results were representative of three independent experiments. 6 BioMed Research International The results indicate that compound 9i activates caspase cascade and finally results in apoptosis.

Compound 9i Induces Mitochondrial Pathway-Mediated
Apoptosis. Apoptosis is caused by a lot of pathways in cells. To clarify which pathway is involved in compound 9i-induced apoptosis, we first checked the mitochondrial pathway activity after 9i treatment. We examined whether the mitochondrial membrane integrity was damaged by compound 9i treatment, and rhodamine 123 was employed to measure the mitochondrial membrane potential (ΔΨm) in the compound 9i-treated Hela cells [18]. The results reveal that the cells with high ΔΨm decrease significantly as the concentration of 9i increased (Figures 5(a) and 5(b)). It means that the mitochondrial pathway is activated in compound 9i-induced apoptosis.
There are a lot of proteins involved in mitochondrial pathway apoptosis, and to determine whether compound 9i-induced apoptosis is associated with B-cell/lymphoma 2 (Bcl-2) family proteins, the expression of proapoptotic (Bax, Bak, Bad, and Bim) and antiapoptotic (Bcl-2 and Bcl-xL) proteins was measured by western blot. The expression of proapoptotic proteins was significantly increased as compound 9i concentration increased, while the expression of antiapoptotic proteins Bcl-2 and Bcl-xL was decreased ( Figure 5(c)). These results reveal that compound 9i triggers ΔΨm collapse, proapoptotic protein activation, and caspase protein activation and finally results in apoptosis. Taken together, compound 9i induces mitochondrial pathwaymediated apoptosis.
Cervical cancer is a huge threat to women, while there is a great shortage of chemotherapeutic molecules for advanced cervical cancer [19]. Imidazopyridines are one type of nitrogenous heterocycles which have the ability to influence many cellular pathways for proper functioning of cancer cells, immune system, and enzymes involved in carbohydrate metabolism, etc. [20]. The imidazopyridine analogue O4I3 inhibits histone lysine demethylase 5 (KDM5) activity to promote reprogramming of resistant fibroblasts [21]. Numerous imidazopyridines have a prominent anticancer property in several human cancer cell lines [4,5,22]. Here in the current study, we explored the anticancer effect of novel imidazopyridine compound 9i on the human cervical cancer cell line HeLa. The MTT assay showed that compound 9i suppressed HeLa cell growth in a concentration-and time-dependent manner. Compound 9i inhibited HeLa cell growth with IC 50 of 10.62 μM. Compound 9i also inhibited HeLa cells' clonogenicity. All these data indicate that compound 9i exhibits prominent anticancer properties in human cervical cancer.
To uncover the molecular mechanism of compound 9i inhibiting HeLa cell growth, we checked the cell death of HeLa cells by PI staining. The results showed that HeLa cells were undergoing cell death, which were stained with PI after compound 9i treatment. In addition, the PI-positive cells' chromatin condensed which is a characteristic phenomenon of apoptosis. Apoptosis is the most common mechanism in anticancer drug-induced cell death. The compound 9iinduced apoptosis was demonstrated by FACS with PI/an-nexin V staining and western blot of caspase proteins. To dissect which pathway was involved in compound 9i-induced apoptosis, the mitochondrial membrane potential was measured by FACS. The results indicated that compound 9i induced MOMP in HeLa cells. Proapoptotic protein and antiapoptotic protein expression was measured by western blot, and compound 9i induced downregulation of antiapoptotic proteins and upregulation of proapoptotic proteins. Taken together, compound 9i induced mitochondrial pathway-mediated apoptosis in HeLa cells.
Our study revealed that compound 9i was a cytotoxic reagent in HeLa cells and induced mitochondrial pathwaymediated apoptosis. However, these results were in cultured human cervical cancer cells and should be further confirmed in vivo, such as xenograft human tumor in mice. Since a cytotoxic reagent is a strategy to treat cervical cancer and many clinical trials are still undergoing, compound 9i deserves further investigation to implement its anticervical cancer potential.
Some imidazopyridine derivatives have selectivity to many therapeutic targets for cancer therapy [23]. Two 2,6disubstituted imidazo [4,5-b] pyridine compounds have high activity and selectivity against receptor tyrosine kinases AXL and MER in vitro [24]. Two new bisindole-imidazopyridine hybrids induce apoptosis in a human lung cancer cell line (A549) [25]. A new type of imidazopyridine compound selectively inhibits mTOR and exerts a strong antiproliferative effect against HeLa and NCM460 cell lines [26]. An apoptosis pathway is a typical pathway for targeted cancer therapy, a recent study found that Bcl-2 is a prominent target for cancer treatment [27], and many small molecules were designed to target Bcl-2 [28]. Our study found that compound 9i significantly decreased Bcl-2 expression, and whether compound 9i targeted Bcl-2 or other molecules in the apoptosis pathway needs further exploration.

Conclusions
To draw a conclusion, compound 9i showed high cytotoxicity on cervical cancer cells. The molecular mechanism behind its cytotoxicity is that 9i induces MOMP, activates caspase cascade, and finally results in apoptosis. Hence, compound 9i could be further exploited as a potential lead compound in human cervical cancer therapy.

Data Availability
No data was used to support this study.

Conflicts of Interest
The authors declare no conflict of interest.