Skip to main content
NCBI home page
Cell Proliferation logoLink to Cell Proliferation
. 2015 Oct 26;48(6):718–728. doi: 10.1111/cpr.12226

SL4, a chalcone‐based compound, induces apoptosis in human cancer cells by activation of the ROS/MAPK signalling pathway

L‐H Wang 1, H‐H Li 1, M Li 1, S Wang 1, X‐R Jiang 1, Y Li 1, G‐F Ping 1, Q Cao 1, X Liu 1, W‐H Fang 2, G‐L Chen 2, J‐Y Yang 1, C‐F Wu 1,✉
PMCID: PMC6496741  PMID: 26500049

Abstract

Objectives

SL4, a chalcone‐based compound, exhibits clearly inhibitory effects on HIF‐1 and has been shown to effectively suppress tumour invasion and angiogenesis in vitro and in vivo. Here, studies were conducted to determine SL4's anti‐apoptotic effects and its underlying mechanisms, in human cancer cells.

Materials and methods

Cytotoxicity, apoptotic induction and its involved mechanisms of SL4 were investigated using normal cells, cancer cells and mouse xenograft models. The role of reactive oxygen species (ROS) and mitogen‐activated protein kinase (MAPK) signalling in SL4‐induced apoptosis was explored by manipulating specific scavenger or signalling inhibitors, in cultured cells.

Results

SL4 significantly inhibited cell population growth of human cancer cell lines but exhibited lower cytotoxicity against normal cells. In addition, SL4 effectively induced apoptosis of Hep3B and MDA‐MB‐435 cells by activating procaspase‐8, ‐9 and ‐3, and down‐regulating expression levels of XIAP, but did not affect HIF‐1 apoptosis‐related targets, Survivin and Bcl‐XL. Further study showed that SL4 also reduced mitochondrial membrane potential and promoted generation of ROS. ROS generation and apoptotic induction by SL4 were blocked by NAC, a scavenger of ROS, suggesting SL4‐induced apoptosis via ROS accumulation. We also found that MAPKs, JNK and p38, but not ERK1/2, to be critical mediators in SL4‐induced apoptosis. SP600125 and SB203580, specific inhibitors of JNK kinase and p38 kinase, significantly retarded apoptosis induced by SL4. Moreover, anti‐oxidant NAC blocked activation of JNK and p38 induced by SL4, indicating that ROS may act as upstream signalling of JNK and p38 activation. It is noteworthy that animal studies revealed dramatic reduction (49%) in tumour volume after 11 days SL4 treatment.

Conclusions

These data demonstrate that SL4 induced apoptosis in human cancer cells through activation of the ROS/MAPK signalling pathway, suggesting that it may be a novel lead compound, as a cancer drug candidate, with polypharmacological characteristics.

Introduction

Carcinogenesis is a multi‐step process which includes sustained proliferative signalling, evasion of growth suppression, resistance to cell death, replicative immortality and angiogenesis as well as cell invasion and metastasis 1, 2. The complexity of cancer has led to recent interest in polypharmacological approaches for developing anti‐tumour drugs 3, 4.

Apoptosis, or programmed cell death, plays a vital role in regulation of carcinogenesis 5; thus, apoptotic pathways are attractive targets for development of new anti‐cancer drugs 5, 6. In the apoptotic process, initial stress‐induced damage does not kill cells directly, rather it triggers an apoptotic signalling program that leads to cell death. Reactive oxygen species (ROS) appear to be an important regulatory signal 7, 8. Mechanistically, ROS‐induced apoptosis is mediated directly through the mitochondria and involves opening of permeability transition pore complexes followed by release of cytochrome c into the cytosol, which triggers a caspase cascade culminating in cell death 7, 8. Thus, drugs that promote ROS release in cancer cells may prove to be valuable anti‐cancer therapeutics 9. Notably, it has recently been reported that ROS are also able to promote metastasis and angiogenesis by activating hypoxia‐inducible factor 1 (HIF‐1) signalling 10, 11, 12. Thus, polypharmacological strategies should be considered, using ROS/apoptosis induction‐based anti‐cancer approaches.

Chalcones are plant essential intermediate compounds, in flavonoid biosynthesis 13. Many studies have demonstrated anti‐neoplastic activity of chalcones in multiple tumour cell types 13, 14, 15. In our previous study, we found a novel chalcone‐based compound SL4(5d) which exhibited clearly inhibitory effects on HIF‐1 with clear anti‐invasive and anti‐angiogenic potential 16. Interestingly, work of other groups has demonstrated that chalcone‐based compounds also induce apoptosis by triggering ROS accumulation in a variety of malignant cells 17, 18, 19. Singh and collaborators demonstrated that a novel coumarin–chalcone hybrid induced apoptosis through accumulation of ROS and activation of caspases, in cervical cancer cells 17, and Wu and colleagues showed similar results in liver cancer cells using a novel chalcone compound, millepachine 18. In addition, Champelovier et al. found chalcone‐induced apoptosis by ROS accumulation, but in a caspase‐independent manner 19. The above results directed us to wonder whether SL4 also would promote ROS release and subsequent apoptotic induction. To validate this hypothesis, we investigated effects of SL4 on apoptosis and explored its underlying mechanisms in vitro and in vivo.

Materials and methods

Reagents

SL4[(E)‐1‐(5‐hydroxy‐2,2‐dimethyl‐2H‐chromen‐6‐yl)‐3‐(4‐trifluoromethylphenyl)‐propenone], more than 98% pure, was synthesized in the Medicine Chemistry Laboratory at Shenyang Pharmaceutical University (see Fig. 1a). It was dissolved by DMSO to 100 mm and stored at −20 °C. MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide) was purchased from Sigma (St. Louis, MI, USA) and was dissolved in PBS. Propidium iodide (PI) was purchased from Biosharp (Hefei City, Anhui Province, China) and was dissolved in distilled water. SB203580 (a specific inhibitor of MAPK/p38), SP600125 (a specific inhibitor of MAPK/JNK), PD98095 (a specific inhibitor of MAPK/ERK) and carbonil cyanide 3‐cholorophenylhydrazone (CCCP, an uncoupler of oxidative phosphorylation mitochondria) were bought from Sigma and dissolved in DMSO. Primary antibodies against PARP, caspase‐3, caspase‐8, caspase‐9, ERK1/2, phospho‐ERK1/2, p38, phospho‐p38, JNK and phospho‐JNK were purchased from Cell Signaling Technology (Danvers, MA); antibodies to β‐actin, Bcl‐2, Bax, Bcl‐XL, Survivin and X‐IAP were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Figure 1.

Figure 1

Open in a new tab

Effect of SL4 on human cancer cells and normal cells. (a) Chemical structure of SL4. (b) Growth curve of human cancer cells and human normal cells after being treated with SL4 (0.1, 1, 5, 20, 100 μm) for 72 h.

Cell lines and culture

Human liver cancer cell line Hep3B, human breast cancer cell line MDA‐MB‐435 and human normal mammary epithelial cell line MCF‐10A were obtained from the American Type Culture Collection (Manassas, VA). They were routinely cultured in Dulbecco's modified Eagle's medium (high glucose) supplemented with 10% foetal bovine serum (FBS) and maintained at 37 °C in a humidified incubator, with 5% CO2. Primary human umbilical vascular endothelial cells (HUVECs) were purchased from Life Technology (Carlsbad, CA, USA). HUVECs were cultured in Roswell Park Memorial Institute 1640 medium with 10% foetal bovine serum. Human primary peripheral blood lymphocytes (PBL) were isolated as described previously 20. In brief, following diluting blood with PBS, lymphocytes were isolated by centrifugation for 15 min at 280 g. Cells were washed twice in PBS then suspended in complete RPMI 1640 with 10% foetal bovine serum.

Cell viability assay

In vitro cell viability effects of SL4 were determined by MTT assay 21. In brief, cells (1 × 105 cells/ml) were seeded into 96‐well culture plates. After overnight incubation, they were treated with the variety of concentrations of agents for 72 h. 10 μl 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) solution (2.5 mg/ml in PBS) was then added to each well, and plates were incubated for an additional 4 h at 37 °C. After centrifugation (1000 g, 10 min), medium with MTT was aspirated away, followed by addition of 100 μl DMSO. Optical density of each well was measured at 570 nm using a SpectraMax Paradigm Reader (Molecular Devices, Silicon Valley, CA, USA).

Flow cytometric analysis

Flow cytometric analysis was performed 22. Briefly, 1–5 × 106 human normal cells (HUVEC and MCF‐10A) and human cancer cells (Hep3B and MDA‐MB‐435) were harvested at room temperature after pre‐treatment with the variety of reagents for 48 h. Supernatant was removed, and cells were trypsinized, then ice‐cold 70% ethanol was added. Then the cells were re‐suspended in PBS containing 0.1 mg/ml RNase, and incubated at 37 °C for 30 min. Pellets were suspended in 1.0 ml of 40 μg/ml propidium iodide (PI) and analysed by flow cytometry (Becton Dickinson, San Jose, CA). Cell cycle distribution was measured according to standard procedures, and percentages of cells in the different cell cycle phases (G0/G1, S, or G2/M phase) were calculated using CELLQuest (Becton Dickinson) software. Sub‐G1 peak cells were considered as being apoptotic.

Western blot analysis

1 × 107 Hep3B and MDA‐MB‐435 cells were gathered after pre‐treatment for 48 h. Western blotting was performed. In brief, an equal amount of total protein extracts from cultured cells or tissues were fractionated using 10–15% SDS‐PAGE and electrically transferred to polyvinylidene difluoride (PVDF) membranes. Mouse or rabbit primary antibodies and horseradish peroxidase (HRP)‐conjugated appropriate secondary antibodies were used to detect the designated proteins. Bound secondary antibodies on PVDF membranes were reacted with ECL detection reagents (Thermo Scientific, Waltham, MA, USA) and exposed in the dark room. Results were normalized to β‐actin internal control.

Mitochondrial membrane potential assay

Mitochondrial membranes were monitored using rhodamine 123(Rh123) fluorescent dye (Ex/Em = 507/529), which is selectively taken up by mitochondria proportional to mitochondrial membrane potential 20. In brief, cells treated with SL4 (6, 12, 24 μm) or CCCP (10 μm) were collected and washed in PBS, then incubated with 2.5 μg/ml Rh123 in the dark for 30 min at 37 °C. They were then washed in PBS and analysed by flow cytometry (Becton Dickinson).

Examination of intracellular ROS accumulation

Intracellular hydrogen peroxide levels were monitored by flow cytometry, after staining with DCFH‐DA (6‐carboxy‐2’,7’‐dichlorodihydrofluorescein diacetate (Molecular Probes, Eugene, OR). Briefly, cells in the logarithmic growth phase (1 × 105 cells/ml in 35 mm polystyrene culture dishes) were treated with selected concentrations of SL4 for 48 h, then labelled with 10 μmol/l DCFH‐DA for 1 h. Next, they were trypsinized, and washed in PBS, then analysed by flow cytometry (Becton Dickinson). Percentages of cells displaying increased dye uptake were used to reflect increase in ROS levels.

In vivo anti‐tumour efficacy

To determine in vivo anti‐tumour activity of SL4, viable human breast cancer MDA‐MB‐435 cells (1 × 107/100 μl PBS per mouse) were subcutaneously (s.c.) injected into the right flank of 7‐ to 8‐week‐old female Balb/c nude mice. Cell numbers were confirmed by trypan blue staining prior to injection. When average tumour volumes reached 100 mm3, mice were randomly divided into various treatment and control groups (six mice per group). Body weight was recorded once every 2 days. After 2 weeks, mice were sacrificed and tumours were weighed.

TUNEL assay

Tumour tissues embedded in paraffin wax were cut into 4 μm sections, deparaffinized and treated with citrate buffer. The terminal deoxynucleotide transferase‐mediated dUTP nick end labelling (TUNEL) system (Roche, Basel, Switzerland) was used to detect apoptosis in the sections, according to the manufacturer's protocol. TUNEL reaction solution was substituted with TdT‐free solution to provide negative controls. Sections were pre‐treated 10 min with DNase and visualized after diaminobenzidine (DAB) staining. Positive nuclei were identified by their brown coloration.

Statistical analysis

Statistical analysis was performed using spss 11.5 software package for Windows (SPSS, Chicago, IL). Data are presented as mean ± SEM. Statistical significance was calculated using Student's t‐test, with probability level of P < 0.05 considered to be statistically significant.

Results

SL4 selectively inhibited viability of the human cancer cells

The MTT method was used to measure SL4 inhibiting viability of the human liver cancer cell line (Hep3B), human breast cancer cell line (MDA‐MB‐435), human umbilical vein endothelial cells (HUVEC), human immortalized mammary epithelial cell line (MCF‐10A) and human primary peripheral blood lymphocytes (PBL), after 72 h continuous treatment. As shown in Fig. 1b, Hep3B and MDA‐MB‐435 cell treatment with SL4 (0.1–100 μm) led to significant reduction in cell viability, with IC50 values of 6.1 ± 1.2 and 8.4 ± 1.5 μm, respectively. IC50 values of normal human cells (MCF‐10A, HUVEC and PBL) were 16.9 ± 2.4, 17.2 ± 3.5 and 226.0 ± 9.9 μm respectively. Mean IC50 value for normal cells was 86.7 μm, 11.9‐fold lower than that of cancer cells. These data suggest that SL4 is more cytotoxic to human malignant cells and less cytotoxic to normal human cells.

SL4 selectively induced apoptosis of human cancer cells

To determine whether reduction of cell viability of human cancer cells induced by SL4 was associated with apoptosis, Hep3B and MDA‐MB‐435 cells were treated with SL4 at different concentrations. Flow cytometric data showed that percentages of apoptotic Hep3B cells treated with SL4 increased from 20.2 to 56.1% with concentration increasing from 6 to 24 μm at 48 h, whereas percentage of apoptotic cells was just 0.8% in the vehicle control (Fig. 2a). Similarly, treatment of MDA‐MB‐435 cells with SL4 for 48 h also clearly induced concentration‐dependent apoptosis (Fig. 2a). To further confirm the above data, PARP cleavage, a further marker of apoptosis, was determined by western blotting. Figure 2b shows that PARP cleavage increased in a concentration‐dependent manner, in both cancer cell lines. To further explore the underlying mechanisms mediating selectivity of SL4 to human cancer cells and normal cells, we also detected apoptosis‐inducing effect of SL4 on HUVEC and MCF‐10A human normal cells. Compared to the cancer cells, SL4 treatment still resulted in concentration‐dependent increase in apoptosis in human normal cells, but at lower levels (Fig. S1). Taken together, these results suggest that SL4 killed human cancer cells by selectively inducing apoptosis of human cancer cells.

Figure 2.

Figure 2

Open in a new tab

SL4‐induced apoptosis in Hep3B and MDA‐MB‐435 cells. Hep3B and MDA‐MB‐435 cells were treated with a variety of concentrations of SL4 (6, 12, 24 μm) for 48 h. (a) Apoptosis was assessed by flow cytometric analysis. These experiments were repeated in duplicate. Data are presented as the means. (b) Apoptosis was assessed by western blotting.

SL4 activated caspases and down‐regulated XIAP in human cancer cells

As apoptosis is usually associated with activation of caspase‐cascades 23, we further investigated involvement of caspases in SL4‐induced apoptosis. First, we measured caspase‐3, considered as being a key effector caspase, in apoptosis 23. After treatment of Hep3B and MDA‐MB‐435 with SL4 for 48 h, obvious cleavage of procaspase‐3 was revealed along with increasing concentration (Fig. 3). Additionally, activities of initiator caspases (caspase‐8, ‐9) were also assessed. As shown in Fig. 3, treatment with SL4 resulted in enhanced cleavages of procaspase‐8 and procaspase‐9 in both cell lines, compared to those in the vehicle control. These results indicate that SL4 induced cell death by stimulating caspase‐dependent apoptotic pathways.

Figure 3.

Figure 3

Open in a new tab

Effect of SL4 on apoptosis‐related proteins of Hep3B and MDA‐MB‐435 cells. Hep3B and MDA‐MB‐435 cells were treated with SL4 as mentioned above. Then protein extracts were immunoblotted with specific antibodies to caspase‐3, caspase‐8, caspase‐9, Bcl‐XL, Bcl‐2, Bax, Survivin and XIAP.

To further explore the underlying molecular mechanisms of apoptosis induced by SL4, we evaluated expression levels of Bax, Bcl‐2, Bcl‐XL, Survivin and XIAP proteins by western blotting. Of these apoptosis‐related proteins, Bcl‐XL, and Survivin have been reported to be targets of HIF‐1 signalling 24, 25. Thus, we first assessed effects of SL4 on the two proteins in both Hep3B and MDA‐MB‐435 cells. Results indicated that SL4 treatment had no obvious effects on expression of Bcl‐XL or Survivin (Fig. 3), suggesting HIF‐1 signalling is not involved in SL4‐induced apoptosis. Similarly, our results showed that SL4 had no significant effect on expression of either Bax or Bcl‐2. Alternatively, XIAP was clearly down‐regulated by SL4 in both cell lines (Fig. 3). These results demonstrate that XIAP, but not Bax, Bcl‐2, Bcl‐XL or Survivin, were also involved in SL4‐induced apoptosis in the cancer cells.

SL4‐induced apoptosis through the ROS/mitochondria pathway

XIAP and its mediated inhibition of procaspase‐9 are important events of the mitochondria‐apoptosis pathway resistance of cancer cells 26. Activation of procaspase‐9 and inhibition of XIAP by SL4 suggest SL4‐induced apoptosis might be via the mitochondrial apoptotic pathway. To verify whether SL4 induced loss of mitochondrial membrane potential (MMP), we used rhodamine 123 to detect its alterations in Hep3B and MDA‐MB‐435 cells. As shown in Fig. 4a, after staining with rhodamine 123, green fluorescence decreased in SL4‐treated Hep3B cells in a concentration‐dependent manner, indicating loss of MMP. This effect was also evident after exposure of MDA‐MB‐435 cells to SL4 (Fig. 4a).

Figure 4.

Figure 4

Open in a new tab

Effect of SL4 on mitochondrial membrane potential ( MMP ) and reactive oxygen species ( ROS ) generation. Hep3B and MDA‐MB‐435 cells were treated with SL4 as mentioned above. (a) MMP release was assessed by flow cytometric analysis. Cells were labelled with 2.5 μg/ml Rh123 in the dark for 30 min followed by treatment with SL4. CCCP was used as positive control. (b) ROS release was assessed by flow cytometry. Cells were labelled with 10 μmol/l DCFH‐DA for 1 h prior to treatment. (c) Apoptosis was measured by flow cytometry.

Mitochondria are the major source of ROS and ROS can determine the fate of cancer cells by regulating the mitochondrial apoptosis pathway 27. To determine whether ability of SL4‐induced apoptosis was mediated by ROS, we used flow cytometry to measure intracellular ROS in Hep3B and MDA‐MB‐435 cells. Cells were treated with the chosen concentrations of SL4 for 48 h, and DCF fluorescence was recorded as a measure of intracellular ROS levels. As shown in Fig. 4b, levels of intracellular ROS had significant and concentration‐dependent increases in both Hep3B and MDA‐MB‐435 cells. To confirm that ROS generation was responsible for SL4‐induced apoptosis, cells were pre‐treated with NAC (10 mm), an inhibitor of ROS production, for 4 h followed by incubation with SL4 (24 μm). We found that pre‐treatment with NAC not only inhibited generation of ROS (Fig. 4b) but also reduced apoptosis induced by SL4 in MDA‐MB‐435 cells (Fig. 4c). Overall, these results suggest that the ROS/mitochondria pathway mediates SL4‐induced apoptosis.

SL4‐induced apoptosis by activation of the MAPK pathway

To investigate possible roles of the MAPK pathway in SL4‐induced apoptosis, we assessed levels of MAPKs (JNK, p38, and ERK1/2) in SL4‐treated Hep3B and MDA‐MB‐435 cells. As shown in Fig. 5a, we found that exposure of either line to SL4 resulted in activation of JNK and p38 (phosphorylation). Expression of JNK and p38 (unphosphorylated form) were not altered by SL4 treatment, but on the other hand, we did not observe activation of ERK1/2 in MDA‐MB‐435 cells exposed to varying concentrations of SL4. In contrast, exposure of Hep3B cells to SL4 led to activation of ERK1/2 (Fig. 5a). This difference may be due to dissimilar cell types.

Figure 5.

Figure 5

Open in a new tab

Effect of SL4 on the MAPKs pathway. Hep3B and MDA‐MB‐435 cells were treated with SL4 as mentioned above. (a) Expression of JNK, phosphorylated JNK, p38, phosphorylated p38, ERK1/2 and phosphorylated ERK1/2 was detected by western blotting. (b) Apoptosis was assessed by western blotting. (c) Expression of JNK, phosphorylated JNK, p38 and phosphorylated p38 were detected by western blotting. Cells were pre‐treated with NAC (10 mm) for 4 h and incubated with SL4 (24 μm) for 48 h.

To further verify the role of MAPKs in SL4‐induced apoptosis, Hep3B or MDA‐MB‐435 cells were pre‐treated for 4 h with a specific inhibitor for JNK, SP600125(10 μm), a specific inhibitor for p38, SB203580(10 μm), or a specific inhibitor of ERK1/2, PD98095(10 μm). Inhibitor‐treated cells were exposed to SL4 for 48 h, and then apoptosis was determined by western blotting. As shown in Fig. 5b, cleavage of PARP was significantly enhanced in cultures exposed to 24 μm SL4 compared to vehicle control. However, this effect was blocked by JNK inhibitor SP600125 in both cell lines. Similarly, pre‐treatment with specific p38 inhibitor SB203580 also led to marked reduction of SL4‐ caused PARP cleavage in MDA‐MB‐435 cells. However, co‐treatment of Hep3B cells with ERK1/2 inhibitor PD98095 and SL4 did not result in reduction in PARP cleavage compared to that in PD98095 single treated cells. These data strongly suggest that MAPK/JNK and MAPK/p38 pathways play an important role in SL4‐induced apoptosis.

SL4‐induced activation of MAPKs was dependent on ROS generation

In view of simultaneous occurrence and interaction between ROS generation and MAPK activation 28, we next investigated which event was the primary factor. Our data showed that pre‐treatment with ROS scavenger NAC blocked activation of JNK and p38 induced by SL4 in MDA‐MB‐435 cells (see Fig. 5c), results suggesting ROS to be upstream signalling for activation of JNK and p38.

SL4 inhibited xenograft growth in nude mice by inducing apoptosis

To further investigate in vivo tumour growth and SL4 treatment, MDA‐MB‐435 tumours were implanted into Balb/C mice. Our data showed that SL4 administration caused significant inhibitory effects on growth of MDA‐MB‐435 tumours. Finally, mean tumour volume in mice of the SL4‐treated groups (2.5 and 5 mg/kg, i.v.) were 470 ± 69 and 313± 75 mm3, respectively, compared to 614 ± 88 mm3 for the control group (Fig. 6a). Inhibition by SL4 at dose of 5 mg/kg was 49.0%. Furthermore, we found that body weights of SL4‐treated groups showed no significant loss compared to mice of the control group (Fig. 6b). Taken together, these results demonstrated that SL4 was an effective anti‐tumour lead compound. Moreover, we also assessed apoptosis in tumour tissues by TUNEL staining. Immunohistochemical data showed that, compared to vehicle‐treated tumours, administration of SL4 resulted in enhancement of apoptosis in MDA‐MB‐435 tumours (Fig. 6c).

Figure 6.

Figure 6

Open in a new tab

Anti‐tumour effect of SL4 on MDA‐MB‐435 human xenograft models. (a) Mice transplanted with MDA‐MB‐435 human xenografts were randomly divided into three groups and injected with SL4 (2.5, 5 mg/kg/day, i.v.) or vehicle, for a period of 11 days. Tumour volumes are expressed as mean ± SD (n = 4–6 per group). (b) Body weight of MDA‐MB‐435 human xenografted mice after administration with the variety of doses of SL4. (c) Apoptosis was revealed by immunohistochemistry using a TUNEL kit in MDA‐MB‐435 xenografted tumour tissues. Quantification of effects of SL4 on apoptosis is shown in column diagram. All error bars are SEM. *Denotes significant difference compared to control, P < 0.05.

Discussion

In this study, our results for the first time illustrated that SL4, a novel chalcone‐based compound, selectively killed tumour cells by inducing apoptosis, shown by activation of caspase‐3, caspase‐8, caspase‐9 and PARP. Moreover, we found that SL4 reduced tumour burdens in MDA‐MB‐435 xenografted mice without gross toxicity. Importantly, we revealed underlying mechanisms of apoptotic induction, involving promotion of ROS release, subsequent activation of the MAPK signalling pathways, followed by loss of mitochondrial membrane potential (MMP).

The caspase family of cysteine proteases plays important roles in both initiation and execution of apoptosis. Initiator caspase‐8 and caspase‐9 mainly contribute to activation of the death receptor pathway and the mitochondrial apoptosis pathway, respectively. However, executioner caspase‐3 is responsible for cleaving its substrates, such as poly ADP‐ribose polymerase (PARP), subsequently inducing apoptosis 29, 30. In the study, our data showed that treatment with SL4 resulted in enhanced cleavage of procaspase‐8, procaspase‐9, procaspase‐3 and PARP in both Hep3B and MDA‐MB‐435 cell lines, suggesting that SL4 induced cell death by stimulating caspase‐dependent apoptotic pathways.

Inhibitors of apoptosis (IAP) proteins are endogenous inhibitors of apoptosis 31, and the best characterized of these are XIAP and Survivin, which inhibit caspase‐9 and caspase‐3 activation, respectively, thereby negatively regulating apoptosis 26, 32. Our results showed that SL4 reduced expression of XIAP, which might partially explain the reason of activation of caspase‐9 induced by SL4. In contrast, there was no change in Survivin after treatment with SL4, indicating that activation of caspase‐3 by SL4 was mediated by upstream signalling.

Apoptosis regulator Bcl‐2 is a member of the family of evolutionarily related proteins, which govern MMP and can be either pro‐apoptotic, such as Bax, or anti‐apoptotic such as and Bcl‐2, and Bcl‐XL 33. In this study, levels of anti‐apoptotic proteins Bcl‐2 and Bcl‐XL, were not altered significantly in Hep3B and MDA‐MB‐435 cells after treatment with SL4. Similarly, expression of pro‐apoptotic protein Bax was unchanged following compound treatment. These data suggest that Bcl‐2 family members are unlikely to play a significant role in SL4‐induced apoptosis in these malignant tumour cells.

Although Bcl‐2 family members were not altered after SL4 treatment, activation of caspase‐9 and down‐regulation of XIAP suggested that the mitochondrial apoptosis pathway might play a crucial role in SL4‐induced apoptosis. Integrity of MMP is a key checkpoint of the mitochondrial apoptosis pathway 34. We observed significant reduction of MMP after treatment with SL4, demonstrating that the mitochondrial apoptosis pathway is involved in apoptosis‐induced by SL4. ROS is a cellular metabolite which regulates multiple cancer‐related signalling pathways. Increased levels of ROS may result in apoptosis by oxidizing mitochondrial pores, thereby disrupting MMP 7, 8. In this study, we found that levels of intracellular ROS underwent significant and concentration‐dependent increases both in Hep3B and MDA‐MB‐435 cells, when treated with SL4. This is consistent with our data concerning concentration‐dependent decline in mitochondrial membrane potential. In addition, pre‐treatment with NAC, an inhibitor of ROS production, not only completely inhibited generation of ROS but also partially suppressed apoptosis induced by SL4 in MDA‐MB‐435 cells. Overall, these results suggest that SL4 induced apoptosis, at least partially by the ROS‐mediated mitochondrial apoptotic pathway.

Mitogen‐activated protein kinases, including ERK, p38 and JNK, mediate intracellular signal transduction in response to different extracellular stimuli, resulting in regulation of gene expression implicated in a wide array of physiological processes, including apoptosis 35, 36. Numerous studies have demonstrated that activation of JNK and p38 are involved in apoptosis induced by various stimuli or anti‐cancer drugs, such as arsenic trioxide 37, taxel 38, cisplatin 39 and vinblastine 40. Consistent with these previous studies, our results indicate that SL4 significantly increased phosphorylation of JNK and p38 in both Hep3B and MDA‐MB‐435 cells. The role of JNK and p38 was further supported by results that induction of apoptosis by SL4 was partially reversed by pre‐treatment with SP600125 (JNK inhibitor) and SB203580 (p38 inhibitor). In contrast to JNK and p38, the role of ERK1/2 in drug‐induced apoptosis is controversial 36. Our results showed that SL4 treatment led to activation of p‐ERK1/2 in Hep3B cells. However, pre‐treatment with PD98095 (ERK1/2 inhibitor) did not reverse apoptosis induced by SL4. This result suggests that ERK1/2 might not play a crucial role in SL4‐induced apoptosis. Taken together, these results demonstrate that SL4 induced apoptosis by activating phosphorylation of JNK and p38 in these cancer cells.

It has been reported that ROS is able to activate JNK and p38 kinase to induce apoptosis 41. However, recent studies elucidated that inactivation of JNK and p38 by specific inhibitors also results in inhibition of ROS release 42, 43, indicating that JNK or p38 might also regulate ROS to some extent. Here our data showed that pre‐treatment with ROS scavenger NAC blocked activation of JNK and p38 induced by SL4 in MDA‐MB‐435 cells, suggesting that ROS may be an upstream signal for activation of JNK and p38.

In conclusion, this study showed that a novel chalcone, SL4, inhibited tumour growth by inducing apoptosis in vitro and in vivo. Mechanistically, SL4 activated caspases and reduced XIAP, involved in both the death‐receptor apoptosis pathway and mitochondrial apoptotic pathway. One the other hand, SL4 stimulated the ROS‐mediated mitochondrial apoptotic pathway to induce apoptosis in the tumour cells (Fig. 7), and SL4 has been identified as an HIF‐1 inhibitor with anti‐invasive and anti‐angiogenic potential 16. SL4 may be a novel lead compound as a cancer drug candidate with polypharmacological properties.

Figure 7.

Figure 7

Open in a new tab

Depiction of potential anti‐tumour mechanisms of SL4. SL4 inhibited tumour invasion and angiogenesis via block HIF‐1 signalling. In contrast, SL4 activated caspases 3, 8, 9 and reduced XIAP. Simultaneously, SL4 stimulated ROS‐mediated‐MAPKs/mitochondrial apoptotic pathways to induce apoptosis in tumour cells.

Supporting information

Fig. S1 SL4‐induced apoptosis in HUVEC and MCF‐10A cells HUVEC and MCF‐10A cells were treated with the appropriate concentrations of SL4 (6, 12, 24 μm) for 48 h and apoptosis was assessed by flow cytometry.

Click here for additional data file. (95KB, ppt)

Acknowledgements

The authors gratefully acknowledge the National High Technology Research and Development Program of China (863 Program) (no. 2012AA020305), Excellent Talents Plan in Liaoning Province (no. LJQ2014111), China Postdoctoral Science Foundation funded project (no. 20100481213, 2012T50474) and Shenyang Pharmaceutical University Science Foundation (no. ZCJJ2013401) for their financial support.

References

  • 1. Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144, 646–674. [DOI] [PubMed] [Google Scholar]
  • 2. Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell 100, 57–70. [DOI] [PubMed] [Google Scholar]
  • 3. Dar AC, Das TK, Shokat KM, Cagan RL (2012) Chemical genetic discovery of targets and anti‐targets for cancer polypharmacology. Nature 486, 80–84. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4. Knight ZA, Lin H, Shokat KM (2010) Targeting the cancer kinome through polypharmacology. Nat. Rev. Cancer 10, 130–137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5. Dixon SC, Soriano BJ, Lush RM, Borner MM, Figg WD (1997) Apoptosis: its role in the development of malignancies and its potential as a novel therapeutic target. Ann. Pharmacother. 31, 76–82. [DOI] [PubMed] [Google Scholar]
  • 6. Hickman JA (1992) Apoptosis induced by anticancer drugs. Cancer Metastasis Rev. 11, 121–139. [DOI] [PubMed] [Google Scholar]
  • 7. Afanas'ev I (2011) Reactive oxygen species signaling in cancer: comparison with aging. Aging Dis. 2, 219–230. [PMC free article] [PubMed] [Google Scholar]
  • 8. Matés JM, Segura JA, Alonso FJ, Márquez J (2012) Oxidative stress in apoptosis and cancer: an update. Arch. Toxicol. 86, 1649–1665. [DOI] [PubMed] [Google Scholar]
  • 9. Ivanova D, Bakalova R, Lazarova D, Gadjeva V, Zhelev Z (2013) The impact of reactive oxygen species on anticancer therapeutic strategies. Adv. Clin. Exp. Med. 22, 899–908. [PubMed] [Google Scholar]
  • 10. Wang Y, Ma J, Shen H, Wang C, Sun Y, Howell SB et al (2014) Reactive oxygen species promote ovarian cancer progression via the HIF‐1α/LOX/E‐cadherin pathway. Oncol. Rep. 32, 2150–2158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11. Kim J, Koyanagi T, Mochly‐Rosen D (2011) PKCδ activation mediates angiogenesis via NADPH oxidase activity in PC‐3 prostate cancer cells. Prostate 71, 946–954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12. López‐Lázaro M (2007) Dual role of hydrogen peroxide in cancer: possible relevance to cancer chemoprevention and therapy. Cancer Lett. 252, 1–8. [DOI] [PubMed] [Google Scholar]
  • 13. Winter E, Chiaradia LD, de Cordova CA, Nunes RJ, Yunes RA, Creczynski‐Pasa TB (2010) Naphthylchalcones induce apoptosis and caspase activation in a leukemia cell line: the relationship between mitochondrial damage, oxidative stress, and cell death. Bioorg. Med. Chem. 18, 8026–8034. [DOI] [PubMed] [Google Scholar]
  • 14. Winter E, Chiaradia LD, Silva AH, Nunes RJ, Yunes RA, Creczynski‐Pasa TB (2014) Involvement of extrinsic and intrinsic apoptotic pathways together with endoplasmic reticulum stress in cell death induced by naphthylchalcones in a leukemic cell line: advantages of multi‐target action. Toxicol. In Vitro 28, 769–777. [DOI] [PubMed] [Google Scholar]
  • 15. Winter E, Devantier Neuenfeldt P, Chiaradia‐Delatorre LD, Gauthier C, Yunes RA, Nunes RJ et al (2014) Symmetric bis‐chalcones as a new type of breast cancer resistance protein inhibitors with a mechanism different from that of chromones. J. Med. Chem. 57, 2930–2941. [DOI] [PubMed] [Google Scholar]
  • 16. Wang LH, Chen GL, Lu XH, Wang S, Han SH, Li Y et al (2015) Novel chalcone derivatives as hypoxia‐inducible factor (HIF)‐1 inhibitor: synthesis, anti‐invasive and anti‐angiogenic properties. Eur. J. Med. Chem. 89, 88–97. [DOI] [PubMed] [Google Scholar]
  • 17. Wu W, Ye H, Wan L, Han X, Wang G, Hu J et al (2013) Millepachine, a novel chalcone, induces G2/M arrest by inhibiting CDK1 activity and causing apoptosis via ROS‐mitochondrial apoptotic pathway in human hepatocarcinoma cells in vitro and in vivo. Carcinogenesis 34, 1636–1643. [DOI] [PubMed] [Google Scholar]
  • 18. Singh N, Sarkar J, Sashidhara KV, Ali S, Sinha S (2014) Anti‐tumour activity of a novel coumarin‐chalcone hybrid is mediated through intrinsic apoptotic pathway by inducing PUMA and altering Bax/Bcl‐2 ratio. Apoptosis 19, 1017–1028. [DOI] [PubMed] [Google Scholar]
  • 19. Champelovier P, Chauchet X, Hazane‐Puch F, Vergnaud S, Garrel C, Laporte F et al (2013) Cellular and molecular mechanisms activating the cell death processes by chalcones: critical structural effects. Toxicol. In Vitro 27, 2305–2315. [DOI] [PubMed] [Google Scholar]
  • 20. Zhang H, Yang JY, Zhou F, Wang LH, Zhang W, Sha S et al (2011) Seed oil of brucea javanica induces apoptotic death of acute myeloid leukemia cells via both the death receptors and the mitochondrial‐related pathways. Evid. Based Complement. Alternat. Med. 2011, 965016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21. Wang LH, Yang JY, Yang SN, Li Y, Ping GF, Hou Y et al (2014) Suppression of NF‐κB signaling and P‐glycoprotein function by gambogic acid synergistically potentiates adriamycin‐induced apoptosis in lung cancer. Curr. Cancer Drug Targets 14, 91–103. [DOI] [PubMed] [Google Scholar]
  • 22. Wang LH, Li Y, Yang SN, Wang FY, Hou Y, Cui W et al (2014) Gambogic acid synergistically potentiates cisplatin‐induced apoptosis in non‐small‐cell lung cancer through suppressing NF‐κB and MAPK/HO‐1 signalling. Br. J. Cancer 110, 341–352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23. Shi Y (2004) Caspase activation: revisiting the induced proximity model. Cell 117, 855–858. [DOI] [PubMed] [Google Scholar]
  • 24. Chen N, Chen X, Huang R, Zeng H, Gong J, Meng W et al (2009) BCL‐xL is a target gene regulated by hypoxia‐inducible factor‐1{alpha}. J. Biol. Chem. 284, 10004–10012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25. Li W, Chen YQ, Shen YB, Shu HM, Wang XJ, Zhao CL et al (2013) HIF‐1α knockdown by miRNA decreases survivin expression and inhibits A549 cell growth in vitro and in vivo. Int. J. Mol. Med. 32, 271–280. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Obexer P, Ausserlechner MJ (2014) X‐linked inhibitor of apoptosis protein – a critical death resistance regulator and therapeutic target for personalized cancer therapy. Front. Oncol. 4, 197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27. Figueira TR, Barros MH, Camargo AA, Castilho RF, Ferreira JC, Kowaltowski AJ et al (2013) Mitochondria as a source of reactive oxygen and nitrogen species: from molecular mechanisms to human health. Antioxid. Redox Signal. 18, 2029–2074. [DOI] [PubMed] [Google Scholar]
  • 28. Son Y, Kim S, Chung HT, Pae HO (2013) Reactive oxygen species in the activation of MAP kinases. Methods Enzymol. 528, 27–48. [DOI] [PubMed] [Google Scholar]
  • 29. Green DR (2000) Apoptotic pathways: paper wraps stone blunts scissors. Cell 102, 1–4. [DOI] [PubMed] [Google Scholar]
  • 30. Pop C, Salvesen GS (2009) Human caspases: activation, specificity, and regulation. J. Biol. Chem. 284, 21777–21781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31. Schimmer AD (2004) Inhibitor of apoptosis proteins: translating basic knowledge into clinical practice. Cancer Res. 64, 7183–7190. [DOI] [PubMed] [Google Scholar]
  • 32. Tamm I, Wang Y, Sausville E, Scudiero DA, Vigna N, Oltersdorf T et al (1998) IAP‐family protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95), Bax, caspases, and anticancer drugs. Cancer Res. 58, 5315–5320. [PubMed] [Google Scholar]
  • 33. Lindsay J, Esposti MD (1813) Gilmore AP (2011) Bcl‐2 proteins and mitochondria – specificity in membrane targeting for death. Biochim. Biophys. Acta 4, 532–539. [DOI] [PubMed] [Google Scholar]
  • 34. Susin SA, Zamzami N, Kroemer G (1998) Mitochondria as regulators of apoptosis: doubt no more. Biochim. Biophys. Acta 1366, 151–165. [DOI] [PubMed] [Google Scholar]
  • 35. Wagner EF, Nebreda AR (2009) Signal integration by JNK and p38 MAPK pathways in cancer development. Nat. Rev. Cancer 9, 537–549. [DOI] [PubMed] [Google Scholar]
  • 36. Deschênes‐Simard X, Kottakis F, Meloche S, Ferbeyre G (2014) ERKs in cancer: friends or foes? Cancer Res. 74, 412–419. [DOI] [PubMed] [Google Scholar]
  • 37. Kang YH, Lee SJ (2008) The role of p38 MAPK and JNK in Arsenic trioxide‐induced mitochondrial cell death in human cervical cancer cells. J. Cell. Physiol. 217, 23–33. [DOI] [PubMed] [Google Scholar]
  • 38. Selimovic D, Hassan M, Haikel Y, Hengge UR (2008) Taxol‐induced mitochondrial stress in melanoma cells is mediated by activation of c‐Jun N‐terminal kinase (JNK) and p38 pathways via uncoupling protein 2. Cell. Signal. 20, 311–322. [DOI] [PubMed] [Google Scholar]
  • 39. Brozovic A, Osmak M (2008) Activation of mitogen‐activated protein kinases by cisplatin and their role in cisplatin‐resistance. Cancer Lett. 251, 1–16. [DOI] [PubMed] [Google Scholar]
  • 40. Selimovic D, Badura HE, El‐Khattouti A, Soell M, Porzig BB, Spernger A et al (2013) Vinblastine‐induced apoptosis of melanoma cells is mediated by Ras homologous A protein (Rho A) via mitochondrial and non‐mitochondrial‐dependent mechanisms. Apoptosis 18, 980–997. [DOI] [PubMed] [Google Scholar]
  • 41. Pan JS, Hong MZ, Ren JL (2013) Reactive oxygen species: a double‐edged sword in oncogenesis. World J. Gastroenterol. 15, 1702–1707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42. Li W, Wu Z, Ma Q, Liu J, Xu Q, Han L et al (2013) Hyperglycemia regulates TXNIP/TRX/ROS axis via p38 MAPK and ERK pathways in pancreatic cancer. Curr. Cancer Drug Targets 14, 348–356. [DOI] [PubMed] [Google Scholar]
  • 43. Zeng KW, Song FJ, Wang YH, Li N, Yu Q, Liao LX et al (2014) Induction of hepatoma carcinoma cell apoptosis through activation of the JNK‐nicotinamide adenine dinucleotide phosphate (NADPH) oxidase‐ROS self‐driven death signal circuit. Cancer Lett. 353, 220–231. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1 SL4‐induced apoptosis in HUVEC and MCF‐10A cells HUVEC and MCF‐10A cells were treated with the appropriate concentrations of SL4 (6, 12, 24 μm) for 48 h and apoptosis was assessed by flow cytometry.

Click here for additional data file. (95KB, ppt)

Articles from Cell Proliferation are provided here courtesy of Wiley

RESOURCES