Apoptosis is Induced in Cancer Cells via the Mitochondrial Pathway by the Novel Xylocydine-Derived Compound JRS-15

The novel compound JRS-15 was obtained through the chemical modification of xylocydine. JRS-15 exhibited much stronger cytotoxic and pro-apoptotic activity than its parent compound in various cancer cell lines, with IC50 values in HeLa, HepG2, SK-HEP-1, PC-3M and A549 cells ranging from 12.42 to 28.25 μM. In addition, it is more potent for killing cancer than non-cancerous cells. Mechanistic studies showed that JRS-15 treatment arrested cell cycle at the G1/S phase, which further triggered the translocation of Bax and Bak to the mitochondria, resulting in mitochondrial membrane potential (MMP) depolarization and the subsequent release of cytochrome c and the second mitochondria-derived activator of caspase (Smac). The sequential activation of caspase-9 and caspase-3/7 and the cleavage of poly (ADP-ribose) polymerase (PARP) were observed following these mitochondrial events. Caspase-8, an initiator caspase that is required to activate the membrane receptor-mediated extrinsic apoptosis pathway was not activated in JRS-15-treated cells. Further analysis showed that the levels of the anti-apoptotic proteins Bcl-xL and XIAP were significantly reduced upon JRS-15 treatment. Furthermore, the caspase-9 inhibitor z-LEHD-fmk, the pan-caspase inhibitor z-VAD-fmk, and Bcl-xL or XIAP overexpression all effectively prevented JRS-15-induced apoptosis. Taken together, these results indicate that JRS-15 induces cancer cell apoptosis by regulating multiple apoptosis-related proteins, and this compound may therefore be a good candidate reagent for anticancer therapy.

we show that JRS-15, functions as a broad-range anticancer agent by effectively triggering apoptosis than xylocydine. Mechanistic studies in HeLa cells demonstrate that JRS-15 triggers the intrinsic apoptosis pathway.
JRS-15 appeared as a solid powder, white in color; this is totally different from the original yellow color of xylocydine. In addition, both of these two compounds are highly soluble in DMSO.

JRS-15 Inhibits the Growth of Various Cancer Cells
To examine whether JRS-15 has a cytotoxic effect on human cancer cells, we assessed the viability of five different human cancer cell lines after treatment with JRS-15 using the MTT assay. The control groups were treated with 0.1% (v/v) DMSO, and no differences were found between 0.1% (v/v) DMSO and the negative controls. As shown in Figure 2 and Table 1, JRS-15 exhibited a broad-spectrum of growth inhibition and cytotoxicity against these cancer cell lines. JRS-15 strongly inhibited the growth of HeLa, HepG2 and A549 cells, with IC 50 values ranging from 12.42 to 14.25 μM. It had moderate cytotoxic activity in SK-HEP-1 and PC-3M cells, with IC 50 values of approximately 28.00 μM. Furthermore, the cytotoxic activity of JRS-15 was much greater than that of its parent compound xylocydine. Importantly, the variation in IC 50 value of JRS-15 against different cell lines indicates that JRS-15 is more potent to kill cancer than non-cancerous cells.

JRS-15 Induces Apoptotic Cell Death in Various Cancer Cells
To examine whether JRS-15 decreased cell viability by inducing apoptosis, we analyzed the cells for morphological characteristics typical of apoptosis. Following 24 h of treatment with 25 μM JRS-15, over 82.4% of HeLa cells demonstrated characteristic apoptotic morphology, such as cell rounding and membrane blebbing ( Figure 3A, top middle). In addition, DAPI staining showed that JRS-15 induced both chromatin condensation and nuclear fragmentation ( Figure 3A, bottom middle). These morphological changes were dramatically blocked by pre-treatment of the cells with a pan-caspase inhibitor, z-VAD-fmk ( Figure 3A, right panel). We then analyzed the cell cycle progression of HeLa cells after 24 h of treatment with 25 μM JRS-15. The fraction of cells with sub-G1 DNA content increased to 49.36% after JRS-15 treatment. In contrast, only 0.33% of cells in the control group had sub-G1 DNA content ( Figure 3C). Similar effects were detected by measuring the fraction of Annexin V-FITC/PI-positive cells using flow cytometry. The result showed that over 12.26% early apoptosis and 49.19% late apoptosis appeared after cells treated with 25 μM JRS-15 for 24 h ( Figure 3D). We then measured the cell-free caspase-3 activity in five cancer cell lines (HeLa, HepG2, SK-HEP-1, PC-3M and A549 cells) treated with 25 μM JRS-15 for 24 h. The results showed that the caspase-3 activities of the treated cells increased 1.99-to 8.79-fold in comparison to the control groups ( Figure 3E). The effect of JRS-15 on caspase-3 activity in these cells was further evaluated by immunoblotting analysis to measure PARP (a specific substrate of caspase-3) cleavage, and similar effects on PARP cleavage were observed under these conditions ( Figure 3F). However, the caspase-3 activities were not detected upon xylocydine treatment for 24 h, even at a concentration of 50 μM, except for HepG2 cells with slight elevation ( Figure 3E,F). Similar effects were observed in HeLa cells upon xylocydine (75 μM, time gradient) treatment by kinetic study and immunoblot analysis on caspase-8, -9, and PARP ( Figure S1). So in the following analysis on the dynamics of apoptosis induced by JRS-15, we didn't perform the assays with xylocydine as a comparison. Importantly, the caspase-3 activity was not detected under 25 μM JRS-15 or 50 μM xylocydine treatment in LO2 cells ( Figure 3E,F). In addition, the cleavages of caspase-8, -9 and PARP in LO2 cells were not observed, even at a concentration of 30 μM JRS-15 treatment for 24 h ( Figure S2). In conclusion, JRS-15 induced cell death in multiple cancer cell lines by triggering apoptosis in a caspase-dependent manner. JRS-15 is more powerful than xylocydine, and exhibited selective at inducing apoptosis between cancer and normal liver cells.

JRS-15-Induced Apoptosis in HeLa Cells is Caspase-9-Dependent
To identify the caspase activation pathway in JRS-15-induced HeLa cell apoptosis, we investigated the activation kinetics of the initiator caspases-8 and -9 and the downstream effector caspase-3. The results showed that caspase-9 activity was slightly elevated in cells that had been treated with JRS-15 for 12 h. The activities of caspase-9 and caspase-3 were markedly upregulated after cells were treated with JRS-15 for 18 h and 24 h, whereas the caspase-8 activity remained unchanged at 24 h of treatment ( Figure 4A). Similarly, immunoblot analysis demonstrated that caspase-9 and PARP cleavage was increased in a time-dependent manner when cells were treated with JRS-15 for 18 h or longer. However, no proteolytic activation of caspase-8 was detected ( Figure 4B). Moreover, in HeLa cells, JRS-15-induced morphological changes were significantly blocked by pre-treatment with z-LEHD-fmk, a caspase-9 inhibitor, or z-VAD-fmk, a pan-caspase inhibitor ( Figure 4C). Similarly, PARP cleavage was also inhibited in the cells pre-treated with caspase-9 or pan-caspase inhibitor but not in cells treated with z-IETD-fmk, a caspase-8 inhibitor ( Figure 4D). These observations demonstrate that JRS-15-induced HeLa cell apoptosis depends on caspase-9 and caspase-3 activation.

JRS-15 Triggers the Mitochondrial Apoptotic Pathway in HeLa Cells
Cytochrome c and Smac release from the mitochondrial inner membrane space into the cytosol has been shown to be a key event in the activation of caspase-9, which subsequently initiates a caspase cascade involving caspase-3 [9,13]. Because JRS-15-induced HeLa cell apoptosis is mediated by the caspase-9 and caspase-3 pathway (Figure 4), we investigated cytochrome c and Smac release in JRS-15-treated HeLa cells. Immunoblot analysis demonstrated that cytochrome c and Smac in the cytoplasmic fraction appeared at 12 h after JRS-15 treatment and gradually increased over time ( Figure 5A). The protein levels of cytochrome c and Smac in the whole-cell fraction did not undergo any detectable changes ( Figure 5B).
The Bcl-2 family proteins control MOMP, and therefore cytochrome c and Smac release, while the multi-domain Bcl-2 family members Bax and Bak serve as a necessary gateway for the release of apoptotic factors [17,18]. We assessed the translocation of Bax and Bak by immunoblot analysis. Bax and Bak proteins were detected in HeLa cell mitochondrial fractions after 12 h of treatment with JRS-15, and the levels of these proteins continued to increase in a time-dependent manner ( Figure 5C). This increase was consistent with the data in Figure 5A. In contrast, the levels of Bax and Bak in whole-cell fractions did not change upon JRS-15 treatment ( Figure 5D). (A) Cell-free caspase-3, -8 and -9 activities were measured (* p < 0.01, ** p < 0.001). (B) Whole-cell lysates were analyzed by immunoblotting for caspase-8, caspase-9, PARP and β-actin. HeLa cells were pre-treated or not with 100 μM z-IETD-fmk, a caspase-8 specific inhibitor, z-LEHD-fmk, a caspase-9 specific inhibitor, and z-VAD-fmk, a pan-caspase inhibitor for 2 h before treatment with 25 μM JRS-15 for 24 h. (C) Apoptotic cell death was measured by counting apoptotic cells, and (D) whole-cell lysates were analyzed by immunoblotting for PARP and β-actin.
MMP depolarization assays were also performed. HeLa cells were treated or not with JRS-15 for the indicated times and stained with a mitochondria-specific cation dye (MitoCapture). The results showed that the intensity of bright red fluorescence, indicative of normal mitochondria, decreased at 12 h and diminished significantly at 18 h and 24 h after JRS-15 treatment ( Figure 5E, top). In contrast, the intensity of diffuse green fluorescence, indicative of depolarized mitochondria, was inversely related to that of the red fluorescence in JRS-15-treated cells ( Figure 5E, bottom). The close association of Bax and Bak translocation, MMP depolarization, and cytochrome c and Smac release with subsequent activation of caspase-9 clearly suggests that JRS-15-induced HeLa cell apoptosis occurs through the intrinsic mitochondrial apoptosis pathway. whole-cell lysates were analyzed by immunoblotting for cytochrome c, Smac, Bax, Bak, α-tubulin, β-actin, and COX II. The numbers below the bands of Cyto. c, Smac, Bax and Bak represent each band's relative abundance to the loading controls, which were quantified by Image-Pro Plus software. (E) Cells were stained with a mitochondria-specific cation dye (MitoCapture). Images were captured using a fluorescence microscope with excitation wavelengths of 570 nm and 500 nm, respectively. Cells with normal, polarized mitochondria emit punctate red fluorescence, while depolarized mitochondrial membranes emit a diffuse green fluorescence.

JRS-15 Reduces Bcl-xL and XIAP Protein Levels in HeLa Cells
We measured the levels of anti-apoptotic proteins to further understand the mechanisms underlying JRS-15-induced HeLa cell apoptosis. Immunoblot analysis in HeLa cells demonstrated that the levels of the anti-apoptotic molecules Bcl-xL and XIAP were downregulated at 12 h post-treatment, and their degradation was significantly increased at 18 h and 24 h after JRS-15 treatment. In contrast, the levels of other anti-apoptotic molecules, such as Bcl-2, c-IAP-1 and c-IAP-2, showed no detectable changes ( Figure 6A). To further investigate whether JRS-15-induced HeLa cell apoptosis is due to Bcl-xL and XIAP downregulation, pEGFP-N3/pCS4, pEGFP-N3/pCS4-Bcl-xL, and pEGFP-N3/pCS4-XIAP were co-transfected into HeLa cells for 24 h followed by JRS-15 treatment of the transfected cells for an additional 24 h. At the end of the treatment period, cells with either normal or apoptotic morphology were identified by counting the GFP-positive cells. Our results indicated that following JRS-15 treatment the percentage of apoptotic cells amongst the Bcl-xL-and XIAP-overexpressing cells was significantly lower than in the control group ( Figure 6B,C). Similarly, the caspase-3 activity was markedly attenuated in Bcl-xL-and XIAP-overexpressing cells under these conditions ( Figure 6D). These data suggest that JRS-15-induced HeLa cell apoptosis is associated with the downregulation of anti-apoptotic proteins, Bcl-xL and XIAP. Moreover, overexpression of Bcl-xL and XIAP significantly inhibited JRS-15-induced HeLa cell apoptosis.

JRS-15 Induces Cell Cycle Arrest in HeLa Cells
To gain an insight into the mechanism of the anti-proliferative activity of JRS-15, its effects on cell cycle distribution were determined in HeLa and LO2 cells. The results showed that JRS-15 strongly blocked the cell cycle at G1/S phase in HeLa cells under the low concentrations in a dose-dependent fashion ( Figure 7A,C), whereas minimally influenced the normal liver LO2 cells under the same conditions ( Figure 7B,C). As mentioned above, JRS-15 treatment with 25 μM could induce apoptosis in various cancer cell lines ( Figure 3E,F). These observations suggest that JRS-15-induced HeLa cell apoptosis may start with cell cycle arrest, which triggers further apoptotic signaling.

Low Caspase-9 Expression in SK-HEP-1 Cells May Contribute to Their Resistance to JRS-15-Induced Apoptosis
The data shown in Figure 2 and Table 1 suggest that the IC 50 values of JRS-15 in SK-HEP-1 and PC-3M cells were higher than those in HeLa, HepG2, and A549 cells. In addition, the caspase-3 activity in SK-HEP-1 and PC-3M cells was only slightly elevated compared with that in the other three cell lines following 25 μM JRS-15 of treatment ( Figure 3E). Similar results were obtained for PARP cleavage under the same conditions ( Figure 3F). Interestingly, the IC 50 value of JRS-15 was dramatically decreased, to 16.62 μM, in a SK-HEP-1 cell line that stable overexpressing caspase-9 (SK-Cas-9), and the cell viability trend was similar to that of HepG2 cells ( Figure 8A,B). In addition, caspase-3 activity was significantly increased in SK-Cas-9 cells compared with SK-HEP-1 cells ( Figure 8C). Similar effects were also observed in the PARP cleavage assay after treatment of cells with 25 μM JRS-15 ( Figure 8D). These data suggest that the downregulation of caspase-9 in SK-HEP-1 may underlie the resistance of these cells to JRS-15-induced apoptosis.

Discussion
An increasing number of studies have implicated apoptosis as an important mechanism by which chemotherapeutic agents kill susceptible cells [3]. Previous reports have demonstrated that xylocydine is able to induce apoptosis in HCC cells, but its pro-apoptotic property is weak [26]. Here, we show that JRS-15, a novel compound which is derived from xylocydine by replacing the 6-Br position with 3-(3-bromophenyl)-phenyl (Figure 1), can effectively inhibits the cell growth on multiple cancer cell types by triggering apoptosis, such as cervical, hepatic, prostate and lung cancer cells, and its anti-proliferative effects are more powerful than xylocydine (Figures 2 and 3). Importantly, JRS-15 is less cytotoxic to the normal liver cells than cancerous cells (Figure 2), and is selective at inducing apoptosis between cancer and normal cells ( Figure 3E,F and Figure S2). Moreover, cell cycle analysis indicates that JRS-15 minimally influences normal liver LO2 cells compared to HeLa cells under the same conditions (Figure 7).
To investigate the mechanisms of JRS-15-induced apoptosis, we selected HeLa cells as a model system. Here, we have proven that JRS-15 induces HeLa cell apoptosis by a mechanism that is functionally associated with cell cycle arrest, a series of mitochondrial events and caspase-3 activation. JRS-15 treatment at low concentrations resulted in strong G1/S phase cell cycle arrest (Figure 7).
Under high concentrations of treatment, morphological determination, cell cycle distribution analysis, and Annexin V/PI analysis clearly demonstrated that JRS-15 induces caspase-dependent apoptosis (Figure 3). These observations show that JRS-15 triggered apoptosis may start with strongly blocking cell cycle. Further study showed that JRS-15-induced apoptosis involved the elevation of caspase-9 activity, which was detected at 12 h and that the activities of caspase-9 and caspase-3 were dramatically increased at 18 h after JRS-15 treatment ( Figure 4A). This result was consistent with that of immunoblot analysis of caspase-9 and PARP cleavage under the same conditions ( Figure 4B). As expected, the activation kinetics of initiator caspase-9 and the effector caspase-3 correlate well over time with that of the mitochondrial translocation of Bax and Bak, depolarization of MMP, and subsequent release of cytochrome c and Smac (Figures 4 and 5). On the other hand, the downregulation of anti-apoptotic proteins, Bcl-xL and XIAP over time may contribute to JRS-15-induced stronger apoptosis ( Figure 6). Furthermore, pre-treatment with caspase-9 or a pan-caspase inhibitor or Bcl-xL or XIAP overexpression all effectively prevented JRS-15-induced HeLa cell apoptosis ( Figure 4C,D and Figure 6B-E). In addition, the proteolytic activity of initiator caspase-8, required for the extrinsic apoptotic pathway, was not detected under this apoptotic procession (Figure 4). Taken together, these results clearly demonstrate that JRS-15 induces HeLa cell apoptosis by triggering the intrinsic mitochondria-mediated caspase-9-dependent pathway. A proposed mechanism of JRS-15-induced apoptotic signaling has been illustrated in Figure 9. Although both JRS-15 and xylocydine induce apoptosis via the intrinsic apoptosis pathway, there is still one question why JRS-15 is more potent than its parent xylocydine. Previous report has shown that xylocydine triggers apoptosis in HCC cell by inhibiting the activities of Cdk1, Cdk2, Cdk7 and Cdk9. However, JRS-15 losses the inhibitory effects on these Cdks ( Figure S3), and the exact molecular target of JRS-15-induced apoptosis has not been identified yet in this study. Based on Lipinski's "the rule of 5" and a knowledge-based approach in drug discovery and development, the AlogP value of drug-like between (−0.40) and 5.60 will be ideal [28]. According to this principle, JRS-15 with less polarity (AlogP = 2.85) may be more effective to across the cell membrane than xylocydine (AlogP = −1.53, polar) does, which partially explains the more potent anticancer activity of JRS-15.
Interestingly, the five cancer cell lines tested showed differential sensitivities to JRS-15 ( Figure 2, Table 1). JRS-15 was more effective at inducing apoptosis in HeLa, HepG2, and A549 cells than in SK-HEP-1 and PC-3M cells ( Figure 3E,F). Dysregulation of the apoptosis pathway is a common feature of cancer cells. Different cancer cells have specific expression patterns of apoptosis-regulating proteins [31,32]. In this study, JRS-15 showed quite different cell growth inhibition and pro-apoptotic effects in the two hepatoma cell lines, HepG2 and SK-HEP-1 ( Figure 2, Table 1 and Figure 3E,F). We examined the expression patterns of apoptosis-regulating proteins in these two cell lines [33], and found that the expression level of caspase-9 was much lower in SK-HEP-1 than in HepG2 cells ( Figure 8A). We then constructed a caspase-9 overexpressed SK-HEP-1 cell line (SK-Cas-9), and determined cell viability and caspase-3 activity upon treatment with JRS-15. SK-Cas-9 cells exhibited markedly increased sensitivity to JRS-15 in terms of cell viability and in vitro caspase-3 activity compared with SK-HEP-1 cells, and the viability trend was similar to that in HepG2 cells (Figure 8). These observations suggest that the reduced expression of caspase-9 in SK-HEP-1 cells may partially explain the mechanism of JRS-15 resistance, and restoration of caspase-9 expression is able to reestablish JRS-15-induced SK-HEP-1 cell apoptosis.

Chemistry: General
The 1 H and 13 C NMR data were recorded with a Varian Mercury 300 NMR spectrometer, using TMS as an internal standard. Chemical shifts (δ) are given in parts per million and coupling constants are given as absolute values expressed in Hertz. Mass spectra were obtained using LC/MS 1100 of Agilent Technology Corporation and Alltech ELSD 2000 instrument. Melting points were determined by an X-4 digital microscope. Column chromatography was generally performed on silica gel (300-400 mesh) and TLC inspections on silica gel GF254 plates.
The above obtained compound 12 was treated with NaOMe (16.00 mg, 0.30 mmol) in MeOH (5 mL) for 12 h at room temperature or until completion of the reaction as shown by TLC. The mixture was co-evaporated with silica and chromatographed using

Cell Culture and MTT Assay
HeLa, HepG2, SK-HEP-1, PC-3M, and A549 cells were maintained at 37 °C in 5% CO 2 in Dulbecco's Modified Eagle's Medium (DMEM) (Invitrogen, Carlsbad, CA, USA) supplemented with 10% newborn calf serum (Invitrogen, Carlsbad, CA, USA) and 100 µg/mL penicillin, 100 µg/mL streptomycin (Invitrogen, Carlsbad, CA, USA). LO2 cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen), the other cultured conditions were the same as described above. Cells were seeded in 96-well plates at 1 × 10 4 cells/well in triplicate. After incubation for 24 h, cells were treated with indicated concentrations of JRS-15 or xylocydine. At 44 h post-treatment, 20 µL of MTT (5 mg/mL) was added to each well and incubated for 4 h. The formazan crystals formed by viable cells were solubilized with 200 µL of DMSO, and the color intensity was measured at 550 nm with an ELISA plate reader (BioTec Instruments, Winooski, VT, USA).

DAPI Staining Assay
HeLa cells were cultured for 24 h and treated or not with z-VAD-fmk for 2 h prior to treatment with 25 μM JRS-15 for 24 h. Cells were then fixed and stained with DAPI to visualize the nuclei and DNA. Images were captured under a fluorescence microscope (Olympus, Tokyo, Japan).

Immunoblot Analysis
Cells were washed with ice-cold PBS and solubilized in a RIPA lysis buffer containing a protease inhibitor cocktail (Roche, South San Francisco, CA, USA) and 1 mM PMSF. After incubation on ice for 1 h, the insoluble materials were removed by centrifugation at 12,000 rpm for 15 min. Equivalent amounts of total protein (50 μg or 100 μg) from each sample were analyzed by SDS-PAGE followed by electro-transfer to a PVDF membrane (Gelman, St. Louis, MO, USA). The membrane was blocked with 5% non-fat milk and probed with the indicated antibodies. The blots were washed and incubated with a horseradish peroxidase-coupled secondary antibody (Pierce), followed by detection by ECL assay (Amersham, Piscataway, NJ, USA).

Preparation of Mitochondrial and Cytosolic Protein Extracts
HeLa cells were treated or not with 25 μM JRS-15 for the indicated times. Mitochondrial and cytosolic protein extracts were prepared using a Mitochondria Isolation Kit (Pierce, Rockford, IL, USA) according to the manufacturer's instructions.

Depolarization Assay of Mitochondrial Membrane Potential
HeLa cells were treated or not with 25 μM JRS-15 for the indicated times and incubated with 1.0 µg/mL of MitoCapture cation dye (Calbiochem, La Jolla, CA, USA) at 37 °C for 30 min. Images were captured by a fluorescence microscope (Olympus, Tokyo, Japan) with excitation wavelengths of 500 and 570 nm, respectively.

Transient Transfection Assay
HeLa cells were plated and then transfected using PolyFect Transfection Reagent (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. Cells were co-transfected with 0.5 μg of pEGFP-N3 and 1.5 μg of pCS4 or pCS4-Bcl-xL or pCS4-XIAP for 24 h followed by an additional 24 h of treatment with JRS-15. Images were captured by a fluorescence microscope (Olympus, Tokyo, Japan).

Analysis the Value of AlogP
The AlogP values of JRS-15 and xylocydine were analysis by Accelrys Discovery Studio 2.5 (Accelrys, San Diego, CA, USA).

Conclusions
In conclusion, JRS-15 functions as a broad-spectrum anticancer agent in human cancer cells by blocking cell cycle and further triggering apoptosis via the intrinsic pathway. This leads to the translocation of Bax and Bak to the mitochondria, resulting in the depolarization of MMP and the release of cytochrome c and Smac, which is coincided with the downregulation of Bcl-xL and XIAP, thereby triggering the downstream caspase cascade. In addition, JRS-15 has less cytotoxic effect on normal cells, and is selective at inducing apoptosis between cancer and normal cells. Taken these together, JRS-15 is a potential candidate as a powerful chemotherapeutic agent. Furthermore, investigating the mechanisms of apoptosis resistance in certain cell lines may provide new solutions for cancer therapy.