Suppression of PKCδ/NF-κB Signaling and Apoptosis Induction through Extrinsic/Intrinsic Pathways Are Associated with Magnolol-Inhibited Tumor Progression in Colorectal Cancer In Vitro and In Vivo

Magnolol is one of the hydroxylated biphenyl compounds from the root and stem bark of Magnolia officinalis, which shown to possess anti-colorectal cancer (CRC) effects. However, the regulatory mechanism of magnolol on apoptosis and NF-κB signaling in human CRC has not been elucidated. Thus, we investigated the inhibitory mechanism of magnolol on human and mouse CRC (HT-29 and CT-26) in vitro and in vivo. Results from reporter gene assay indicated that both magnolol and rottlerin (PKCδ inhibitor) reduced the endogenous NF-κB activity. In addition, indolactam V (PKCδ activator)-induced NF-κB signaling was significantly suppressed with both magnolol and rottlerin treatment. Results from Western blotting also indicated that phosphorylation of PKCδ and NF-κB -related proteins involved in tumor progression were effectively decreased by magnolol treatment. The invasion capacity of CRC cells was also attenuated by both magnolol and rottlerin. Furthermore, magnolol triggered Fas/Fas-L mediated extrinsic apoptosis and mitochondria mediated intrinsic apoptosis were validated by flow cytometry. Most importantly, tumor growth in both HT-29 and CT-26 bearing mice were suppressed by magnolol, but no pathologic change was detected in mice kidney, spleen, and liver. As confirmed by immunohistochemistry (IHC) staining from tumor tissue, PKCδ/NF-κB signaling and downstream proteins expression were decreased, while apoptotic proteins expression was increased in the magnolol treated group. According to these results, we suggest that the induction of apoptosis through extrinsic/intrinsic pathways and the blockage of PKCδ/NF-κB signaling are associated with the magnolol-inhibited progression of CRC.


Introduction
Colorectal cancer (CRC) is a commonly diagnosed cancer and the fourth leading cause of cancer death in the world [1]. Epidemiological studies indicated obesity, a lack of dietary fiber intake, low physical activity, and smoking as unfavorable risk factors are associated with the formation of colorectal cancer [2,3]. For the improvement of survival outcomes of patients with CRC, more effective adjuvant therapy has been developed. New treatment approaches, including targeted therapy and

Magnolol Suppressed Tumor Cell Growth, PKC/NF-κB Signaling, Expression of NF-κB Mediated Downstream Proteins in CRC Cells
In Figure 2A, we identified the toxicity effect of magnolol in CT26 and HT29 cells. The IC 50 of magnolol in CT26 and HT29 cells was around 75 µM at 24 h. Next, we identified whether the phosphorylation of PKCδ, ERK, AKT, and NF-κB was altered by magnolol in CRC cells. In both CT26 and HT29 CRC cells, magnolol can effectively dephosphorylate PKCδ, ERK, AKT and NF-κB molecules ( Figure 2B,C). Western blotting quantification results also illustrated the phosphorylation of these molecules was markedly decreased by magnolol by dose depend manner ( Figure 2D,E). Furthermore, we identified the alteration of NF-κB downstream proteins expression after magnolol treatment. As showed in Figure 2F-I, expression of NF-κB downstream proteins including MCL-1, C-FLIP, XIAP, MMP-2, MMP-9, VEGF, uPA, and CyclinD1 were all significantly reduced by magnolol [26][27][28][29]. Taken together, magnolol induced the inhibition of CRC cells proliferation, the suppression of PKC-δ/NF-κB signaling, and decreasing of NF-κB downstream protein expression. In Figure 2A, we identified the toxicity effect of magnolol in CT26 and HT29 cells. The IC50 of magnolol in CT26 and HT29 cells was around 75 μM at 24 h. Next, we identified whether the phosphorylation of PKCδ, ERK, AKT, and NF-κB was altered by magnolol in CRC cells. In both CT26 and HT29 CRC cells, magnolol can effectively dephosphorylate PKCδ, ERK, AKT and NF-κB molecules ( Figure 2B,C). Western blotting quantification results also illustrated the phosphorylation of these molecules was markedly decreased by magnolol by dose depend manner ( Figure 2D,E). Furthermore, we identified the alteration of NF-κB downstream proteins expression after magnolol treatment. As showed in Figure 2F-I, expression of NF-κB downstream proteins including MCL-1, C-FLIP, XIAP, MMP-2, MMP-9, VEGF, uPA, and CyclinD1 were all significantly reduced by magnolol [26][27][28][29]. Taken together, magnolol induced the inhibition of CRC cells proliferation, the suppression of PKC-δ/NF-κB signaling, and decreasing of NF-κB downstream protein expression.

Magnolol Triggered Both Extrinsic and Intrinsic Apoptosis Effect in CRC Cells
We further evaluated the regulatory mechanism of magnolol on apoptosis in CRC cells. Fas/Fas-L, the death receptor and its ligand, mediated apoptosis were both activated by magnolol treatment in CT26 and HT29 cells ( Figure 3A,B). In Figure 3C, cleaved caspase-8 was increased by magnolol. Moreover, magnolol also increased the loss of ΔΨm and the activated caspase-9 ( Figure  3D-E). Subsequently, caspase-3 was activated 20-40 % by the magnolol treatment group. Annexin-V and PI double positive population, defined as late apoptosis and necroptosis ( Figure 3G, I), was also effectively induced by magnolol. In the context of the above results, we suggested that magnolol may trigger both death receptor and mitochondria dependent apoptosis mechanism in CRC cells.

Magnolol Triggered Both Extrinsic and Intrinsic Apoptosis Effect in CRC Cells
We further evaluated the regulatory mechanism of magnolol on apoptosis in CRC cells. Fas/Fas-L, the death receptor and its ligand, mediated apoptosis were both activated by magnolol treatment in CT26 and HT29 cells ( Figure 3A,B). In Figure 3C, cleaved caspase-8 was increased by magnolol. Moreover, magnolol also increased the loss of ∆Ψm and the activated caspase-9 ( Figure 3D,E). Subsequently, caspase-3 was activated 20-40% by the magnolol treatment group. Annexin-V and PI double positive population, defined as late apoptosis and necroptosis ( Figure 3G,I), was also effectively induced by magnolol. In the context of the above results, we suggested that magnolol may trigger both death receptor and mitochondria dependent apoptosis mechanism in CRC cells.

Inhibition of PKCδ/NF-κB Signaling Was Associated with Magnolol-Diminished Invasion Ability of CRC Cells
In addition to apoptosis effect, we further investigated whether inhibition of PKC=δ/NF-κB signaling was implicated with magnolol-abolished invasion ability of CRC cells. The transwell invasion assay was performed in CT26 and HT29 cells after magnolol, Rottlerin, and QNZ treatment. As showed in Figure 4A,C, the number of invasion CT26 cells was reduced by magnolol in a dose dependent manner. Furthermore, after Rottlerin and QNZ treatment the invasion CT26 cells percentage was also significantly decreased. In the meantime, HT29 also showed a similar invasion reduction effect after magnolol, Rottlerin, and QNZ treatments ( Figure 4B,C). These results were

Inhibition of PKCδ/NF-κB Signaling Was Associated with Magnolol-Diminished Invasion Ability of CRC Cells
In addition to apoptosis effect, we further investigated whether inhibition of PKC-δ/NF-κB signaling was implicated with magnolol-abolished invasion ability of CRC cells. The transwell invasion assay was performed in CT26 and HT29 cells after magnolol, Rottlerin, and QNZ treatment. As showed in Figure 4A,C, the number of invasion CT26 cells was reduced by magnolol in a dose dependent manner. Furthermore, after Rottlerin and QNZ treatment the invasion CT26 cells percentage was also significantly decreased. In the meantime, HT29 also showed a similar invasion reduction effect after magnolol, Rottlerin, and QNZ treatments ( Figure 4B,C). These results were corresponded to the reduction of invasion related proteins expression after magnolol treatment in Figure 2F,I. In conclusion, the inhibition of PKC-δ/NF-κB signaling may participate in magnolol-disrupted the invasion capacity of CRC. corresponded to the reduction of invasion related proteins expression after magnolol treatment in Figure 2F,I. In conclusion, the inhibition of PKC=δ/NF-κB signaling may participate in magnolol-disrupted the invasion capacity of CRC.

Magnolol Effectively Suppressed CRC-Bearing Tumor Growth
To confirm the anti-tumor effect of magnolol, we established both CT26 and HT29 bearing animal models. As illustrated in Figure 5A, HT29 or CT26 cells were inoculated into mice right flank and treated with various dose of magnolol for 20 days. The detailed animal experiment procedure is displayed in figure 5A accompanied by a subtitled description. The tumor volume of CT26 and HT29 was markedly decreased by magnolol ( Figure 5B,C). After eight days of treatment, a significant difference between non-treated and magnolol treated groups was found. A CT scan was performed to confirm tumor growth after treatment. Figure 5D,E shows that the CT scanning results from CT26 and HT29 bearing mice all indicated the inhibition of tumor growth on magnolol treatment. With a higher dose of magnolol, a tumor isolated from CT26 and HT29 bearing mice on day 20 revealed the marked shrinkage as compared to other groups ( Figure 5F). Furthermore, the isolated tumor from each group was measured by scale. Results in Figure 5G-H indicated the smallest tumor weight was found in higher dose of magnolol. Then, we further identified whether magnolol treatment may cause the general toxicity or the pathology change on mice kidney, spleen and liver tissue. Body weight recorded from day 0-20 in CT26 and HT29 bearing mice didn't show any noticeable change ( Figure 5I,J). Pathology H&E staining results also didn't indicate any alteration between non-treated and magnolol treated mice ( Figure 5K,L). These results suggested that magnolol may suppress tumor growth without inducing general toxicity in mice.

Magnolol Effectively Suppressed CRC-Bearing Tumor Growth
To confirm the anti-tumor effect of magnolol, we established both CT26 and HT29 bearing animal models. As illustrated in Figure 5A, HT29 or CT26 cells were inoculated into mice right flank and treated with various dose of magnolol for 20 days. The detailed animal experiment procedure is displayed in Figure 5A accompanied by a subtitled description. The tumor volume of CT26 and HT29 was markedly decreased by magnolol ( Figure 5B,C). After eight days of treatment, a significant difference between non-treated and magnolol treated groups was found. A CT scan was performed to confirm tumor growth after treatment. Figure 5D,E shows that the CT scanning results from CT26 and HT29 bearing mice all indicated the inhibition of tumor growth on magnolol treatment. With a higher dose of magnolol, a tumor isolated from CT26 and HT29 bearing mice on day 20 revealed the marked shrinkage as compared to other groups ( Figure 5F). Furthermore, the isolated tumor from each group was measured by scale. Results in Figure 5G-H indicated the smallest tumor weight was found in higher dose of magnolol. Then, we further identified whether magnolol treatment may cause the general toxicity or the pathology change on mice kidney, spleen and liver tissue. Body weight recorded from day 0-20 in CT26 and HT29 bearing mice didn't show any noticeable change ( Figure 5I,J). Pathology H&E staining results also didn't indicate any alteration between non-treated and magnolol treated mice ( Figure 5K,L). These results suggested that magnolol may suppress tumor growth without inducing general toxicity in mice.

Magnolol Suppressed PKCδ/NF-κB Signaling, NF-κB Related Downstream Proteins Expression and Promoted Apoptotic Aroteins Axpression
Finally, we investigated the alteration in expression of PKCδ/NF-κB, NF-κB regulated downstream proteins and apoptotic proteins in CRC tissues by IHC staining after magnolol treatment. The phosphorylation of PKCδ, ERK, AKT, and NF-κB was decreased by magnolol in CT26 and HT29 bearing mice ( Figure 6A-D). In Figure 6E-H, the anti-apoptosis related proteins that regulated by NF-κB such as C-FLIP and MCL-1 were also diminished by magnolol. Additionally, invasion related and angiogenesis related proteins that regulated by NF-κB, such as MMP-9 and VEGF, were also effectively suppressed by magnolol ( Figure 6I-L). Additionally, magnolol may reduce the protein expression of cyclinD1, the proliferation related molecule. In sum, our results elucidated that magnolol may not only suppress PKCδ/NF-κB mediated tumor progression but trigger extrinsic/intrinsic apoptosis related proteins expression ( Figure 6M-P). Death receptor dependent molecules such as Fas, Fas-L, and cleaved caspase-8 were all increased by magnolol. Moreover, the loss of ∆Ψm and the activated caspase-9, both recognized as mitochondria-dependent apoptosis processes or markers, was also increased by magnolol. Cleaved-caspase-3, an apoptosis marker, was also markedly induced by magnolol treatment. In sum, magnolol not only constrained PKCδ/NF-κB signaling, but also triggered apoptotic (cleaved-caspase-3, -8, and -9) proteins expression in CRC tissues.   dependent molecules such as Fas, Fas-L, and cleaved caspase-8 were all increased by magnolol. Moreover, the loss of ΔΨm and the activated caspase-9, both recognized as mitochondria-dependent apoptosis processes or markers, was also increased by magnolol. Cleaved-caspase-3, an apoptosis marker, was also markedly induced by magnolol treatment. In sum, magnolol not only constrained PKCδ/NF-κB signaling, but also triggered apoptotic (cleaved-caspase-3, -8, and -9) proteins expression in CRC tissues.

Discussion
Protein kinase C (PKC), the family of serine/threonine kinases, regulates oncogenic signaling transduction relevant to tumor cell proliferation, survival, and invasion [30,31]. PKC-delta (PKCδ), the PKC isozyme, has been demonstrated to be an upstream modulator of NF-κB which can promote the expression of NF-κB-mediated anti-apoptotic oncogenes cIAP-2 and C-FLIP in colorectal cancer cells [16,31]. Our results showed that PKCδ inhibitor significantly reduced NF-κB activity, whereas AKT or MAPKs inhibitor did not ( Figure 1B). We also found magnolol suppressed PKC activator-induced NF-κB signaling and phosphorylation of PKCδ in CRC cells ( Figure 1C,D). Notably, the phosphorylation of PKCδ was markedly decreased by magnolol treatment in both CT-26 and HT-29 cells (Figure 2B-E). Those results indicated that PKCδ inactivation is required for magnolol-inhibited NF-κB signaling in CRC. In addition to tumor progression, constitutive activation of NF-κB also mediates resistance to chemotherapy or radiotherapy in CRC. Inhibition of NF-κB signaling has been shown to potentiate anti-CRC efficacy of 5-Fluorouracil (5-FU), the chemotherapeutic agent, or radiation [32][33][34]. We suggest the combination of magnolol and 5-Fu or radiation as a novel potential strategy which may offer therapeutic benefits for patients with CRC.
AKT or ERK is the critical component of phosphoinositide 3-kinase (PI3K)/AKT or RAF/mitogen-activated protein/extracellular signal-regulated kinase (MEK)/ERK signaling pathway, respectively. The high expression of both ERK and AKT phosphorylation was required for the progression of CRC and were correlated with a poor prognosis in patients with CRC [35][36][37].

Discussion
Protein kinase C (PKC), the family of serine/threonine kinases, regulates oncogenic signaling transduction relevant to tumor cell proliferation, survival, and invasion [30,31]. PKC-delta (PKCδ), the PKC isozyme, has been demonstrated to be an upstream modulator of NF-κB which can promote the expression of NF-κB-mediated anti-apoptotic oncogenes cIAP-2 and C-FLIP in colorectal cancer cells [16,31]. Our results showed that PKCδ inhibitor significantly reduced NF-κB activity, whereas AKT or MAPKs inhibitor did not ( Figure 1B). We also found magnolol suppressed PKC activator-induced NF-κB signaling and phosphorylation of PKCδ in CRC cells ( Figure 1C,D). Notably, the phosphorylation of PKCδ was markedly decreased by magnolol treatment in both CT-26 and HT-29 cells (Figure 2B-E). Those results indicated that PKCδ inactivation is required for magnolol-inhibited NF-κB signaling in CRC. In addition to tumor progression, constitutive activation of NF-κB also mediates resistance to chemotherapy or radiotherapy in CRC. Inhibition of NF-κB signaling has been shown to potentiate anti-CRC efficacy of 5-Fluorouracil (5-FU), the chemotherapeutic agent, or radiation [32][33][34]. We suggest the combination of magnolol and 5-Fu or radiation as a novel potential strategy which may offer therapeutic benefits for patients with CRC.
AKT or ERK is the critical component of phosphoinositide 3-kinase (PI3K)/AKT or RAF/mitogen-activated protein/extracellular signal-regulated kinase (MEK)/ERK signaling pathway, respectively. The high expression of both ERK and AKT phosphorylation was required for the progression of CRC and were correlated with a poor prognosis in patients with CRC [35][36][37]. Hsu et al. presented magnolol induced p21 expression and cell cycle arrest through upregulation of ERK activation in colon cancer COLO-205 cells (within 60 min) [25]. Thus, the increased phosphorylation of ERK may be involved in magnolol-induced early response in CRC. In our data presented the protein levels of both ERK and AKT phosphorylation were significantly reduced by magnolol treatment in both CT-26 and HT-29 cells (24 h) and tumor tissue ( Figure 2B-E and Figure 6A-D). Hua et al. also reported that Honokiol may reduce the phosphorylation of AKT, ERK, and NF-κB p65 in HT-29 cells [23]. Our results also demonstrate that magnolol decreased the phosphorylation of AKT, ERK, and NF-κB p65 in HT-29 cells ( Figure 2C).
The activity or expression of caspase-3, caspase-9, or FAS was frequently reduced and associated with a poor outcome in CRC patients [38][39][40]. Extrinsic and intrinsic pathway-initiated apoptosis can be halted by the decreased expression of apoptotic proteins and increased expression of anti-apoptotic proteins [41]. MCL-1, XIAP, and C-FLIP are anti-apoptotic proteins which mediate acquired resistance to therapeutic agents or radiotherapy in CRC [42][43][44]. Lin et al. presented magnolol induced apoptosis through extrinsic and intrinsic pathways and inhibited B-cell lymphoma 2 (BCL-2) expression in HCC Hep-G2 cells [45]. According to our results, apoptosis and extrinsic/intrinsic apoptotic signaling transduction (the activation of Fas, FasL, caspase-8 and -9, and the loss of mitochondrial membrane potential) was significantly triggered by magnolol (Figures 3 and 6M-P). Protein levels of MCL-1, C-FLIP, and XIAP were effectively suppressed with magnolol treatment in CRC in vitro and in vivo ( Figure 2F-I and Figure 6E-H).
In conclusion, this study reveals that magnolol induces apoptosis through extrinsic/intrinsic pathways and inhibits NF-κB signaling through PKCδ inactivation in CRC. We suggested that the induction of apoptosis through extrinsic/intrinsic pathways and the suppression of PKCδ/NF-κB signaling are associated with magnolol-inhibited tumor progression in CRC in vitro and in vivo.

Fas and Fas-L Analysis
CT26 and HT29 cells were seeded in a 12-well plate (2 × 10 5 cells/well) overnight and treated with 0, 75, or 100 µM magnolol for 24 h. Cells were then collected by 2000 rpm centrifugation and stained with 100 µL FACS buffer containing 1 µL FAS-FITC and FASL-PE antibodies for 30 min in dark (Thermo Fisher Scientific). The expression patterns of FAS and FAS-L were detected and quantified by NovoCyte flow cytometry and NovoExpress ® software, respectively. All experiments were performed in triplicate and were repeated at least three times.

Invasion Tranwell Assay
Transwell chambers purchased from BD Biosciences (Franklin Lakes, NJ, USA) were coated with 50 µL matrigel one day before invasion assay. CT26 and HT29 cells were seeded in 10-cm plates (3 × 10 6 cells/well) overnight and treated with 0, 75, or 100 µM magnolol, 4 µM Rottlerin or 0.5 µM QNZ for 24 h. After treatment, cell viability was rapidly determined with trypan blue, before 2 × 10 5 viable cells were re-suspended in 300 µL serum-free medium and added to the upper chamber. The lower chamber was filled with 700 µL RPMI-1640 medium containing 10% FBS. After 48 h of migration, cells that invaded transwell membranes were fixed by 4% formaldehyde, stained by 0.5% crystal violet, photographed by microscope (Nikon ECLIPSE Ti-U, Minato City, Tokyo, Japan), and quantified by ImageJ software version 1.50 (National Institutes of Health, Bethesda, MD, USA) [49].

Western Blot
CT26 and HT29 cells were seeded in 10-cm plates (3 × 10 6 cells/well) overnight and treated with 0, 75, or 100 µM magnolol for 24 h. In addition, for PKC activator evaluation, CT26 and HT29 cells were treated with 20 nM Indolactam V for 0.5, 2, or 4 h. Treated cells were collected and lysed by NP-40 lysis buffer containing both 1% protease inhibitor and 1% phosphatase inhibitor mixture II (Sigma-Aldrich). The protein concentration was measured by BCA Protein Assay Kit (Thermo Fisher Scientific). Forty micrograms of protein per group were separated by 6-12% SDS-page and transferred onto polyvinylidene difluoride (PVDF) membranes (EMD Millipore, Bedford, MA, USA) [50]. Immunoreactive proteins were probed and detected by Immobilon Western Chemiluminescent HRP Substrate (Pierce, Rockford, IL, USA) and ChemiDoc-It imaging system (UVP, Upland, CA, USA), respectively. All the protein expression levels were normalized by actin or phospho-protein/total-protein before comparing with the 0 µM magnolol group.

Animal Experiment
Animal experiments were approved by Institutional Animal Care and Use Committee in China Medical University and followed by the guidelines for the use of laboratory animals. Six-week-old BALB/c (n = 12 for each experiment, repeated twice) and Cg-Foxn1 nu /CrlNarl (NUDE) male mice (n = 15 for each experiment, repeated twice) were purchased from National Laboratory Animal Center, Taipei, Taiwan. Ten million CT26 and HT29 cells were subcutaneous injected into mice right flank for growing as tumor ( Figure 5A). Mice were randomly separated into 3 groups after average tumor volume reached 100 mm 3 , included 0 mg/kg magnolol, 50 mg/kg magnolol and 100 mg/kg magnolol. Tumor volumes were recorded by digital caliper every four days and calculated by following formula: tumor volume = length × width 2 × 0.523. Mice body weight was also recorded every four days (from day 0 to 20). Mice were sacrificed on day 20 and tumors were finally extracted, photographed, measured by digital scales and sliced for immunohistochemistry (IHC) staining.

Hematoxylin and Eosin (H&E) Staining
Kidney, liver, and spleen extracted from mice on day 20 were fixed by 4% formaldehyde and embedded paraffin for further slicing. Slicing and H&E staining were performed by bio-check laboratories ltd (Taipei, Taiwan) as followed with regular procedure [51].

Immunohistochemistry (IHC) Staining
In brief, slices were dehydrated with serial decreasing percentage of ethanol and further procedure was followed by manufacturer IHC staining instructions (EMD Millipore, Burlington, MA, USA). Tumor tissue slices were stained by various primary antibodies, including P-PKC delta, P-ERK, P-AKT, P-NF-Kb p65, MMP-9, VEGF, CyclinD1, C-FLIP, MCL-1, cleaved-caspase-3, cleaved-caspase-8, and cleaved-caspase-9. Slides were imaged using a Nikon ECLIPSE Ti-U microscope under 100× magnification. Positive staining signals were finally quantified by ImageJ software [52]. All the protein expression levels were normalized by actin or phospho-protein/total-protein before comparing with the 0 mg/kg magnolol group.

Statistical Analysis
Results are presented as mean ± SD. The significant differences between control and magnolol treatments were analyzed by Student's t-test and one-way ANOVA. p value < 0.05 and p-value < 0.01 were both defined as an indication of statistical significance.