Inhibition on Proteasome β1 Subunit Might Contribute to the Anti-Cancer Effects of Fangchinoline in Human Prostate Cancer Cells

Fangchinoline is a bisbenzylisoquinoline alkaloid isolated from Radix Stephaniae tetrandrae S. Moore. Fangchinoline and its structure analogue, tetrandrine, exhibited direct binding affinity with recombinant human proteasome β1 subunit and also inhibited its activity in vitro. In cultured prostate PC-3 cells and LnCap cells, fangchinoline could dose-dependently inhibit cell proliferation and caspase-like activity of cellular proteasome which was mediated by proteasome β1 subunit. The inhibitive effect of fangchinoline on caspase-like activity of proteasome was also observed in purified human erythrocyte 20S proteasome. In PC-3 cells, fangchinoline induced cell cycle arrest at G0/G1 phase and apoptosis. Treatment of PC-3 tumor-bearing nude mice with fangchinoline inhibited tumor growth, induced apoptosis and also caused decrease in proteasome activities in tumor xenografts. Dose-dependent and time-dependent accumulation of ubiquitinated proteins and important proteasome substrates such as p27, Bax and IκB-α were observed in fangchinoline-treated cells. Over-expression of proteasome β1 subunit by plasmid transfection increased sensitivity of cells to the cytotoxicity of fangchinoline while knockdown of proteasome β1 subunit ameliorated cytotoxicity of fangchinoline in PC-3 cells. Results of the present study suggested that proteasome inhibition was involved in the anti-cancer effects of fangchinoline. Fangchinoline and its structure analogues might be new natural proteasome inhibitors targeting β1 subunit.


Introduction
Proteasome has emerged as an important and effective target for anti-cancer therapy [1]. In our screening of natural proteasome inhibitors, fangchinoline and tetrandrine, two bisbenzylisoquinoline alkaloids isolated from dried root of Stephaniae tetrandrine S. Moore (family,

Cell culture
Human PC-3 prostate cancer cells and human LnCap prostate cancer cells were purchased from Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. PC-3 cells and LnCap cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 units/mL of penicillin, and 100 μg/mL of streptomycin and maintained at 37°C and 5% CO 2 . Fetal bovine serum, RPMI 1640, penicillin and streptomycin were all from Hyclone.

Surface plasmon resonance biosensor analysis
The recombinant human proteasome β1 subunit, product of PSMB6 gene, was expressed as His-Tag proteins using Escherichia coli, and then purified by affinity chromatography as reported in our previous study [32]. The binding affinities of fangchinoline or tetrandrine to recombinant proteasome catalytic β1 subunit, were assayed in vitro using an Biacore 3000 instrument (Biacore AB, Rapsgatan 7, S-754 50 Uppsala, Sweden) as reported before [33]. Briefly, recombinant proteasome β1 subunit protein was immobilized on a CM5 sensor chip as ligand in 11624.5 RU with N-ethyl-N'- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide according to the standard primary amine-coupling procedures, and HBS-EP (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% (v/v) surfactant P20, pH 7.4) was used as the running buffer. Equilibration of the baseline was performed by a continuous flow of HBS-EP through the chip surface for 1 to 2 h. Biacore data were collected at 25°C with HBS-EP as the running buffer at a constant flow of 20 μL/min. Fangchinoline or tetradrine was serially diluted into the running buffer to 0, 2.4, 3.43, 4.90, 7.0, 10 and 20 μM. The samples were injected into the channels at a flow rate of 20 μL/min, followed by washing with the running buffer. The binding responses were recorded continuously in response units (RU) at a frequency of 1Hz as sensorgrams and presented as a function of time. The association (k a ) and dissociation (k d ) rate constants, the equilibrium dissociation constant (KD ¼ k d k a ) were determined by analysis of the sensorgram-curves obtained at different concentrations of the compound by use of BIA evaluation software version 3.1 (Biacore) and the 1:1 Langmuir binding fitting model.

Assay of enzyme activity of recombinant proteasome catalytic β1 subunit
Briefly, catalytic activity of the recombinant human proteasome β1 subunit was assayed by adding recombinant β1 subunit protein (30 μg) to 100 μL of proteasome activity assay buffer (10 mM Tris-HCL, pH 7.8, 5 mM adenosine triphosphate, 0.5 mM dithiothreitol and 5 mM MgCL 2 •6H 2 O) containing 50 μM Z-LLE-AMC in the presence of fangchinoline or tetrandrine at different concentrations or not. The hydrolase activity of the proteasome catalytic β1 subunit could release the fluorogenic AMC component from substrate Z-LLE-AMC and the AMC release was measured after 2 h incubation using a Microplate Reader Bio-Rad 550 with excitation and emission wavelengths of 360 and 465 nm, respectively.

MTT assay of effects of fangchinoline on proliferation of PC-3 cells and LnCap cells
The inhibitive effects of fangchinoline on proliferation of PC-3 cells or LnCap cells was observed by checking cell viability of cells using MTT assay as described before [33]. Briefly, cells were seeded in 96-well plates at a density of 5×10 4 cells/mL for PC-3 cell and 4×10 4 cells/ mL for LnCap cells. After overnight culture, the media were changed into fresh media containing various concentrations of fangchinoline or 0.1% DMSO (solvent control) for 24, 48 h or 72 h. Cell viability was evaluated by measuring the mitochondrial-dependent conversion of the yellow tetrazolium salt MTT to purple formazan crystals by metabolic active cells. The optical density (proportional to the number of live cells) was assessed with a Microplate Reader Bio-Rad 550 at 570 nm. IC 50 value (half-maximal inhibitory concentration) was calculated by the Logit method.

Assay of cellular proteasome activities of PC-3 cells and LnCap cells
Cells were treated with either solvent control (0.1% DMSO), fangchinoline at different concentrations, or 1 μM carfilzomib (positive control) for 24 h at 37˚C. The enzymatic activities of cellular proteasome in cell lysate were measured as reported in our previous paper [32]. Briefly, cells were harvested, washed with PBS and then lysed in proteasome activity assay buffer for 30 min at 4°C. The homogenate was then centrifuged at 12000 × g for 30 min at 4°C. The supernatant was collected as whole cell extract and the protein content in the supernatant was measured with the Bradford reagent. Catalytic activities of cellular proteasome were assayed by adding whole cell extract (containing 30 μg protein) to 100 μL of proteasome activity assay buffer containing 50 μM fluorogenic peptide substrates such as Z-LLE-AMC for detecting C-L activity, Z-ARR-AMC for detecting T-L activity or Suc-LLVY-AMC for detecting CT-L activity, respectively. The release of fluorogenic AMC from the peptide substrates was then measured as described above.

Assay of C-L activity of purified human 20S proteasome
The 20S proteasome assay kit was bought from Enzo Life Sciences, Inc. (Farmingdale, NY 11735, USA). The K m , V max values of the purified human erythrocyte 20S proteasome for fluorigenic peptide substrate Z-LLE-AMC are measured according to the instructions provided by the manufacturer. Inactivation of purified human erythrocyte 20S proteasome by fangchinoline was determined by monitoring the hydrolysis of substrate in the presence of different concentrations of fangcinoline. Assays were performed at 37°C in a reaction buffer (50 μL) containing 50 mM Tris/HCL, pH 7.5, 25 mM KCL, 10 mM NaCL, 1 mM MgCL 2 , 0.1 μg 20S proteasome and Z-LLE-AMC. Reactions were allowed to proceed for 120 min, and fluorescence data were collected every 1 min, and then plotted as a function of time. The apparent dissociation constant, K i app , was determined by non-linear least-fit of the fractional velocity, v i /v 0 , as a function of [I], where v i is the steady state residual activity of the enzyme in the presence of inhibitor (I) and v 0 is the initial velocity in the absence of inhibitor ).

Induction of cell cycle arrest and apoptosis by fangchinoline in PC-3 cells
The induction of cell cycle arrest and apoptosis by fangchinoline in PC-3 cells was observed by using flow cytometric analysis as described in our previous reports [32][33][34]. Briefly, for flow cytometry analysis of cell cycle, cells were collected after treatment, washed with PBS and then fixed with 70% ethanol at 4°C for overnight. After PBS washes, cells were re-suspended in staining buffer (10 mg/mL RNase A and 5 mg/mL propidium iodide in cold PBS) for 30 min in dark at room temperature and then analyzed using FACSCalibur Flow Cytometer. For flow cytometric analysis of apoptosis, Alexa Fluor 488 Annexin V & PI /Dead cell Apoptosis Kit (Invitrogen) was used. Cells were collected after treatment, washed with PBS and then resuspended in binding buffer and then incubated with Annexin V-FITC and propidium iodide for 15 min in dark at room temperature. Then, flow cytometric analysis was conducted and data analysis was performed with CellQuest software.

PC-3 tumor xenograft experiments
Male nude immunodeficient mice Balb/c-nu-nu, aged 4 weeks, were purchased from Shanghai Experimental Animal Center. All procedures involving animals were approved by the Institutional Animal Care and Use Committee of Shanghai Institute of Materia Medica, Chinese Academy of Sciences. On day 0, PC-3 cells (8×10 6 cells) suspended in 0.2 mL of serum-free RPMI 1640 were inoculated s.c. in the right flank of each mouse (six mice per group). 14 days after inoculation, mice were randomly divided into 3 groups and started injection with either vehicle control or fangchinoline at 25 mg/kg (ip., QD) or 50 mg/kg (ip., QD). Tumor sizes were measured every 4 days using calipers and their volumes were calculated using a standard formula: width 2 ×length/2. The relative tumor volume (RTV) was defined as the ratio of the tumor volume at a indicated time and the tumor volume at the start of drug treatment. Body weight was measured daily. On day 30 after inoculation, mice were sacrificed by CO 2 inhalation and tumor xenografts were dissected. To visualize apoptosis in tumor xenografts, TUNEL labeling was conducted to detect apoptotic nuclei using In Situ Death Detection Kit (Roche Molecular Biochemicals). Briefly, tumor xenografts were fixed with 10% formalin solution at room temperature for 24 h and then embedded by paraffin. Specimens were heated for 1h by Water Bath-Slide Drier (LEICA H1 1220, German), incubated with dimethylbenzene for two times (8 min each) and then incubated with 100%, 95%, 90% and 85% ethanol for two times (3 min each). Specimens were then incubated with 0.1M Citrate buffer (pH 6.0) for microwave irradiation. After washing with PBS for two times (3 min each), slices were incubated with reaction buffer (from the In Situ Death Detection Kit) in a humid atmosphere at 37°C for 1 h. Slices were then washed with PBS three times (3 min each) to remove unincorporated fluorescein-deoxyuridine triphosphate. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) 1 μg/mL for 3 min and then washed with PBS for three times (3 min each). Images were captured using a 20X objective (Olympus BX51, Japan) on an epifluorescence microscope. Furthermore, the proteasome activities of tumor xenografts were assayed. Briefly, tumor xenografts were homogenerized in proteasome activity assay buffer using the FastPrep System (MP FastPrep-24, U.S.A), then centrifugation at 4°C for 30 min. The supernatants were then used for assay of proteasome activities using method as described above for analysis of cellular proteasome activities.

Over-expression of proteasome β1 subunit in PC-3 cells by plasmid transfection
Cells with over-expression of proteasome β1 subunit were obtained by transfection with pcDNA3.1 plasmid encoding full-length human PSMB6 cDNA as described before [34]. For plasmid transfection, 5 × 10 5 cells were seeded into each well of a six-well plate until the cells reached approximately 80% confluence after cultured for 24 h. 2.5 μg pcDNA3.1 plasmid DNA encoding PSMB6 or pcDNA3.1 plasmid DNA without PSMB6 (as negative control) was diluted in 250 μL serum-free medium and then incubated for 20 min at room temperature with a mixture of 10 μL Lipofectamine 2000 (Invitrogen) and 400 μL serum-free medium. The resultant complex mixture was then added to each well of the cell culture plate. After 6 h incubation, the complex medium was replaced with fresh minimum essential medium supplemented with 10% (v/v) FBS and let to grow overnight. Then, G418 (Merck) was added to the medium to reach a final concentration equal to the minimum fatal dose (400 μg/mL). The cells were allowed to grow and passage in the presence of G418 for over 2 weeks. Expression of proteasome β1 subunit in the transfected cells was checked using Western blotting assay and compared with that of wild-type cells and negative-control cells. The cytotoxicity of fangchinoline on cells with over-expression of proteasome β1 subunit was then checked using MTT assay as described above and compared with that of wild-type cells and negative-control cells.

Knockdown of proteasome β1 subunit in PC-3 cells by siRNA transfection
Validated siRNAs for PSMB6 (Sigma-Aldrich) were transfected into PC-3 cells by using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's protocol. Scrambled negative control siRNAs (Sigma-Aldrich) were used as negative control. Expression levels of PSMB6 (proteasome β1 subunit) in wide type cells, PSMB6 knockdown cells (cells treated with siRNAs for PSMB6), negative control cells (cells transfected with scrambled negative control siRNAs) were checked using Western blotting assay. To check the influence of proteasome β1 subunit knockdown on cytotoxicity of fangchinoline, cells were transfected with siRNAs for PSMB6 or scrambled negative control siRNAs for 24 h. Then, cells were seeded into 96-well plates at a density of 5 × 103 cells/well and the effects of fangcinoline on cell proliferation were checked using MTT assay as described above. Furthermore, the effects of fangchinoline on markers of apoptosis and autophagy in cells with normal or knockdown of β1 subunit were checked using Western blotting assay as described above. Briefly, after transfected with siRNAs for PSMB6 or scrambled negative control siRNAs for 24 h, cells were treated with fangchinoline at 40 μM for 24 h. Then, the cells were harvested for Western blotting assay. The primary antibodies used were rabbit anti-caspase 3 antibody (1:1000, Cell Signaling Technology, Cat#9662), rabbit anti-PARP antibody (1:1000, Cell Signaling Technology, Cat#9542) and rabbit anti-LC3B antibody (1:1000, Cell Signaling Technology, Cat#2775). The secondary antibody used was HRP-linked goat anti-rabbit IgG (1:1000, Cell Signaling Technology, Cat#7074).

Statistical analysis
Student's t-test was applied to evaluate the differences between treated and control groups. Data were expressed as mean ± SEM and results from at least 3 independent experiments were used for statistical analysis. Asterisks indicate a significant difference (P < 0.05) compared with un-treated control.

Results
Binding affinities and direct inhibitive effects of fangchinoline/tetrandrine on recombinant proteasome β1 subunit Both fangchinoline ( Fig 1C) and tetrandrine (Fig 1D) exhibited direct binding affinity with recombinant human proteasome β1 subunit. As shown in Fig 1C, RU increased with increasing fangchinoline concentration, which indicated that fangchinoline was able to bind to proteasome β1 subunit in a dose-dependent manner. Similar results were observed for tetrandrine ( Fig 1D). The association (k a ), dissociation (k d) and equilibrium dissociation (K D ) constants of fangchinoline or tetrandrine binding to the proteasome β1 subunit were shown in Table 1. The results suggested that fangchinoline and tetrandrine exhibited similar binding affinity to proteasome β1 subunit. Furthermore, both fangchinoline and tetrandrine could directly inhibit the enzyme activity of recombinant proteasome β1 subunit in vitro. As shown in Fig 1E, fangchinoline dose-dependently inhibited the activity of recombinant proteasome β1 subunit. Similar results were observed for tetrandrine ( Fig 1F).
Inhibitive effects of fangchinoline on proliferation and cellular proteasome activities of cancer cells  (Fig 2C). The IC 50 values of fangchinoline in inhibiting cell proliferation of LnCap cells were 36.18±6.33 μM for 24 h, 11.59±0.22 μM for 48 h, and 12.73±0.59 μM for 72 h treatment, respectively. And, fangchinoline also could significantly inhibit the C-L activity of cellular proteasome but did not affect the activities of proteasome T-L and CT-L activities in LnCap cells (Fig 2D).

Inhibitive effects of fangchinoline on C-L activity of purified human 20S proteasome
The hydrolysis curve of β1-specific fluorigenic peptide substrate Z-LLE-AMC at different concentrations by purified human 20S proteasome was shown in Fig 3A. The K m and V max of the purified proteasome was calculated to be 244.2 μM and 9.749X10 -4 nmol/min, respectively. The representative time-dependent hydrolysis curves of Z-LLE-AMC (75 μM) by proteasome Table 1. Biacore assay results of binding affinity between recombinant human proteasome β1 subunit and fangchinoline or tetrandrine.

Compound
Association

Fangchinoline induced cell cycle arrest and apoptosis in PC-3 cells
Representative results of cell cycle analysis were shown in Fig 4A and statistical analysis results were shown in Table 1. Fangchinoline-treated PC-3 cells appeared to accumulate at the G0/G1 phase with a concomitant decrease in the percentage of cells in the S phase. And, fangchinoline also dose-dependently induced apoptosis in PC-3 cells. Representative results of apoptosis analysis were shown in Fig 4B and statistical analysis results were shown in Table 2.   Table. And, results of TUNEL labeling (Fig 5B) showed that apoptosis was induced in PC-3 xenografts of animals treated with fangchinoline.

Proteasome inhibition in xenografts of animals treated with fangchinoline
As shown in Fig 5C, results of proteasome activity assay of the tumor xenografts indicated that proteasome activities of xenografts of fangchinoline-treated groups were decreased compared with that of vehicle control. The decrease in proteasome activity was significant in 50 mg/kg fangchinoline-treated group.

Fangchinoline induced accumulation of ubiquitinated proteins as well as Ub-IκBα, Ub-p27 and Ub-Bax
Proteasome inhibition would cause accumulation of ubiquitinated proteins. As shown in Fig  6A and Fig 6B,  Over-expression of proteasome β1 subunit increased the sensitivity of cells to cytotoxictiy of fangchinoline As shown in Fig 7A, transfection of plasmid encoding proteasome subunit beta type 6 (PSMB6) cDNA caused over-expression of proteasome β1 subunit in PC-3 cells. Results of MTT assay of the inhibitive effects of fangchinoline on proliferation of wild type, negative control and PSMB6-transfected cells showed that cells with over-expression of proteasome β1 subunit were more sensitive to cytotoxictiy of fangchinoline treatment for 24 h (Fig 7B), 48 h (Fig 7C) or 72 h (Fig 7D).

Knockdown of proteasome β1 subunit ameliorated the cytotoxictiy of fangchinoline
As shown in Fig 8A, knockdown PSMB6 expression by siRNA transfection decreased expression of proteasome β1 subunit in PC-3 cells. MTT assay of the effects of fangchinoline on proliferation of negative control and PSMB6-knockdown cells showed that knockdown of proteasome β1 subunit ameliorated proliferation-inhibiting effects of fangchinoline ( Fig 8B). Furthermore, Western blotting assay of makers of apoptosis (caspase-3 and PARP) and autophagy (LC-3B) showed that knockdown of proteasome β1 subunit also ameliorated fangchinoline-induced apoptosis and autophagy (Fig 8C).

Discussion
In the present study, fangchinoline and tetrandrine, bisbenzylisoquinoline alkaloids isolated from Fangji, were demonstrated to have inhibitive effects on proteasome β1 subunit. They could directly bind with recombinant proteasome β1 subunit, inhibit activity of recombinant proteasome β1 subunit in vitro. Proteasome β1, β2 and β5 subunits exert caspase-like (C-L),  Proteasome-Inhibiting Effects of Fangchinoline However, the Ki of fangchinoline, 356.58±30.63 μM, in inhibiting activity of purified 20S proteasome was much higher than the dose needed for inhibiting cellular proteasome activity. The results indicated that the inhibiting effects of fangchinoline on cellular proteasome activity might include both direct effects on catalytic subunits as well as effects on other regulative components of proteasome. The later might deserve further study in the future. The successful use of proteasome inhibitors bortezomib [35] and carfilzomib [36] in clinic suggested the important role of proteasome inhibitors in anti-cancer therapy. Many compounds including natural products such as marizomib (also known as NPI-0052), celastrol,   and EGCG were in clinical or pre-clinical stages [37]. While, both bortezomib and cartizomib were molecules with inhibition mainly on CT-L activity which was mediated by proteasome β5 subunit [38]. Up to now, the role of β1 or β2 subunit in cancer therapy and the possibility of developing molecules with selective inhibition on the C-L or T-L activity of proteasome into new anti-cancer agents are still not clear. In the present study, fangchinoline and tetrandrine were found to be inhibitors specifically act on proteasome β1 subunit but not β2 or β5 subunit. We then further studied the involvement of proteasome inhibition in the anti-cancer effects of fangchinoline. Consistent with previous results [2,11,13], fangchinoline was found to induce G0/G1 phase arrest and apoptosis in PC-3 cells. And, in nude mice inoculated with PC-3 cells, fangchinoline dose-dependently inhibited tumor growth and induced apoptosis in tumor xenografts. Importantly, inhibition of proteasome activities by fangchinoline was also observed in tumor xenografts of fangchinoline-treated animals.
The cell cycle arrest and apoptosis induced by fangchinoline might be resulted from accumulation of proteasome substrates which played important roles in cell cycle progression and apoptosis. Time-dependent and dose-dependent accumulation of ubiquitinated proteins were observed in PC-3 cells treated with fangchinoline. Specifically, fangchinoline induced accumulation of Ub-p27, Ub-Bax and Ub-IκB-α. These results suggested that fangchinoline could inhibit degradation of p27, Bax and IκB-α. p27 could directly inhibit the activities of CyclinE/ Cdk2 which promotes S phase transition, and CyclinD/Cdk4,6 which promotes cell cycle progression in early G1 to late G1 [39]. Therefore, p27 plays a pivotal role in the control of G0/G1 to S phase transition during cell cycle progression. In the present study, the G0/G1 cell cycle arrest induced by fangchinoline might be resulted from the considerable increase in p27 and subsequent negative control of the cell cycle progression through G0/G1-to S-phase [40]. Our results were consistent with previous reports about increased level of p27 and G0/G1 arrest in fangchinoline-treated cells [13,27]. Notably, it had been shown that cell cycle-dependent caspase-like activity that cleaves p27 is the β1 subunit of the 20S proteasome [41]. The specific role of β1 subunit in direct binding and degrading of p27 was further confirmed in a recent study [39]. Our results that finding fangchinoline as a proteasome β1 subunit inhibitor provided explanation for increase of p27 level and G0/G1 arrest in cells treated with fangchinoline. Furthermore, tetrandrine, which was also found to be a proteasome β1 subunit inhibitor in the present study, was also reported to increase p27 level and induce G0/G1 arrest in cancer cells [18-22, 27, 42]. Bax is a well known protein involved in regulation of apoptosis and proteasome inhibitors were reported to induce apoptosis by regulating degradation of Bax [28,[43][44][45]. Increased in Bax level of fangchinoline-treated cells were found in previous reports [2,13,46] as well as in the present study. The effects of fangchinoline on Bax might contribute to apoptosis induced by fangchinoline. IκB-α is a critical regulative factor of NF-κB pathway which plays role in cell proliferation [47,48] as well as inflammatory response [49][50][51]. Inhibition on degradation of IκB-α might be involved in the effects of fangchinoline on cell proliferation. And, the possible involvement of accumulation of Ub-IκB-α in the anti-inflammatory effects of fangchinoline as well as that of Fangji might deserve further study.
In the present study, to confirm the role of proteasome inhibition in cytotoxicity of fangchinoline, both cells with over-expression of proteasome β1 subunit and cells with knockdown of proteasome β1 subunit were used. Cytotoxicity of fangchinoline was found to be enhanced in viability of negative-control cells and cells transfected with siRNAs for PSMB6 after 72 h treatment of fangchinoline at different concentrations. Data were statistical results of three independent experiments. (C) Effects of fangchinoline treatment on markers of apoptosis and autophagy in negative control cells or cells transfected with siRNAs for PSMB6. Showed were representative results of three independent experiments. doi:10.1371/journal.pone.0141681.g008 cells with over-expression of proteasome β1 subunit and ameliorated in cells with knockdown of proteasome β1 subunit. These results suggested that inhibition on proteasome β1 subunit might be involved in the cytotoxicity of fangchinoline. Furthermore, our results showed that knockdown of proteasome β1 subunit not only ameliorated apoptosis but also ameliorated autophagy induced by fanchinoline. Fangchinoline was reported to induce autophagy in other cancer cells [12] and tetrandrine also could induce considerable autophagy [52][53][54]. Since the ubiquitin-proteasome system and autophagy were closely related [55], it would be interesting in the future to further clarify the role of proteasome inhibition in the autophagy-inducing effects of fangchinoline as well as tetrandrine.
In summary, the main finding in the present study is clarification of the contribution of proteasome inhibition to the anti-cancer effects of fangchinoline. By inducing accumulation of proteasome substrates such as p27, Bax, and IκB-α, fangchinoline induced G0/G1 phase arrest and apoptosis in cultured cancer cells and also inhibited growth of tumor xenografts in nude mice. To be noted, fangchinoline was reported to have other activities such as inducing formation and conformational conversion of DNA G-quedruplexes [56] and reversing multidrug resistance by inhibiting P-glycoprotein activity in multidrug resistant human cancer cells [57]. Therefore, proteasome inhibition might be one of the mechanisms of the anti-cancer effects of fangchinoline.
Supporting Information S1