Introduction: Multiple myeloma (MM) is a B plasma cell malignancy currently incurable, and novel therapeutics are needed. Evidences regarding the effect of natural compound schweinfurthins suggest that hematological cancers showed growth inhibitory effects to this family of compounds at single nanomolar concentrations. In this study, we evaluated the cytotoxicity of the schweinfurthin synthetic analog 5′-methylschweinfurthin G (MeSG) in MM cell lines, to better understand the validity of this compound as a therapeutic candidate for further studies in MM. Methods: MeSG toxicity against MM cell lines RPMI-8226, MM.1S, and H-929 was evaluated. Trypan blue exclusion and MTT assays measured cell viability and mitochondrial activity, respectively. Flow cytometry was performed to detect apoptotic mitochondria. Flow cytometry and Western blotting techniques were used to investigate apoptosis and to examine the cell cycle. Western blotting was used to determine AKT activation upon MeSG treatment. Results: We provide evidence that in all MM cells analyzed, MeSG exerts diverse cytotoxic effects. MeSG treatment of MM.1S and H-929, but not in RPMI-8226, causes a loss of mitochondria membrane potential. MeSG causes an arrest in G2/M, especially in RPMI-8226, supported by decreased levels of cyclin-B1 and early increased levels of p21. Finally, there is a diverse response to the MeSG treatment for AKT phosphorylation. MM.1S and H-929 showed a marked decrease in AKT phosphorylation at earlier time points compared to the RPMI-8226 line. Conclusions: MeSG cytotoxicity has been confirmed in all of 3 cell lines studied. Results suggest an early event of increased reactive oxygen species, and/or involvement of cholesterol homeostasis via decreased AKT activation, both of which are currently under investigation.

Multiple myeloma (MM) is the second most frequent hematological cancer worldwide [1]. MM is characterized by enormous molecular heterogeneity that persists at relapse, likely not affected by the chemotherapeutic treatments [2]. In this study, we included different cell lines to shed light on the diverse biological response of MM to the compound used. MM is an incurable plasma cell neoplasm, and despite recent improvements in treatment, the life expectancy at the diagnosis is low, and the quality of life of MM patients is poor [1‒3]. The severe side effects of the treatments are well known including neuropathy, anemia, fatigue, and nausea; in addition, the complications of the progression of the malignancy, such as bone fractures and frequent infections [3], also degrade quality of life. Therefore, effective novel therapies are needed [4].

Our laboratory has an extensive drug development program aimed at a group of compounds with lipid modulating activity called the schweinfurthins, which has been reviewed recently [5, 6]. Novel synthetic schweinfurthins are attractive potential therapeutics because they showed cytotoxicity in several cell lines of different cancers such as breast, renal, melanoma, colon, lung, and leukemia [7]. In cell culture, synthetic schweinfurthins showed a selective cytotoxicity to malignant plasma cells, especially hematological lineages. Previous work in our laboratory showed that the compound 3-deoxyschweinfurthin B had growth inhibitory effects in MM cell lines RPMI-8226 and U266. This study used a readout of MTT assay and also demonstrated synergy when schweinfurthin was given in combination with lovastatin, an HMGCoA reductase inhibitor [8]. However, further study of the mechanism of these effects in diverse MM cell lines has yet to be carried out. More recently, our group showed that schweinfurthin analog 5′-methyl­schweinfurthin G (MeSG) has in vivo activity against chondrosarcoma in a mouse model, when given at a dose of 20 mg/kg for 5-day cycles [9]. This compound is a second-generation synthetic analog with somewhat better stability than 3-deoxyschweinfurthin B. With this research, we aimed to shed light on the effect of MeSG in MM cell lines, to set the stage for in vivo studies in this disease.

The capability of compounds to modulate cell cycle arrest in G2/M is an attractive topic of study in the laboratory. The cell cycle proceeds in consequential steps involving a series of proteins, expressed at specific checkpoints. Blockages at checkpoints can be triggered by exogenous compounds, inducing the cell cycle arrest [10]. One well-studied checkpoint is at the progression between G2 to mitosis (M) phase, with cyclin-B1 in complex with a cyclin-dependent kinase (CDK) will lead the cell from a late G2 to M phase. However, the cell may fail to enter M phase or complete the mitotic phases in response to an anomalous mitotic event such as damaged/unreplicated DNA, aberrant mitotic spindle, and mitochondrial damage [10, 11]. Persistent blockage of the cell cycle triggers cell death [10, 11].

A fundamental signaling pathway that promotes cell survival, especially in MM cells, is represented by phosphatidylinositol 3-kinase (PI3K) and protein kinase B (AKT) [12, 13]. Because of a central role of PI3K/AKT in the progression of MM, several inhibitors of this pathway are in preclinical or clinical trials [13‒15]. A paper from a team at Eisai Inc. showed that schweinfurthin G decreased AKT phosphorylation at Ser273 in lung cancer cell lines A549 and HCC827 by interfering with trans-Golgi network trafficking [16]. Because our previous work as well as 60 cell-line assay data of several schweinfurthin compounds showed very potent cell growth inhibition (GI) in RPMI-8226 cells [8, 17], here, we set out to explore further the mechanisms of MeSG in additional MM cell lines.

The activity of the schweinfurthins has been tested against the NCI-60 panel and showed strong cytotoxicity/GI in several cell lines [6, 7]. Because of opposite sensitivity, 2 cell lines have been extensively assayed with schweinfurthin analogs. The human glioblastoma cell line SF-295 has shown cytotoxicity upon treatment with schweinfurthin, opposite to the resistance shown by the lung epithelial cell line A549 [6]. This is the case with MM cell lines RPMI-8226 and U266, the latter of which is less sensitive [8].

Previous publications on the cytotoxic effect of schweinfurthins have investigated some aspects of cell signaling, but a comprehensive study on the mechanisms of cell death has yet to be carried out. Despite schwein­furthins being discovered late in the last century [18], the mechanism(s) of action is not yet well understood. This, together with the need of novel therapies for MM treatment due to the short expectation of life of MM patients induced us to test schweinfurthins in MM cell lines. Our research aimed to investigate early mechanism(s) of cytotoxicity induced by the schweinfurthin analog MeSG in 3 MM cell lines: H-929 and MM.1S, which are more representative of patient samples, and RPMI-8226, one of the most used line in literature for in vitro studies [19]. The diverse sensitivity of the cell lines examined in this research demonstrates the importance of addressing the clonality of MM and also denotes that MeSG targets specific mechanism(s) that are essential for cell survival in some cell lines but not in others.

Cell Lines and Culture Conditions

NCI-H929 (H-929) (ATCC CRL-9068), MM.1S (ATCC CRL-2974), and RPMI-8226 (ATCC CCL-155) cells were maintained in complete medium antibiotic free as manufacturer instructions. All cultures were incubated at 37°C and 5% CO2. All cells were tested mycoplasma free using the MycoAlertTM Mycoplasma Detection Assay (Lonza, Switzerland).

Cell Proliferation Assays

Trypan Blue Exclusion Assay

Cells were seeded in T25 flasks (Geneese Scientific, San Diego, CA, USA) at the concentration of 5 × 105/mL and treated in time course. Cell suspensions were washed in phosphate-buffered saline (PBS) and resuspended in equal volume of trypan blue (Invitrogen, 0.4% solution in PBS) and were counted using a hemocytometer. Bright field pictures were taken using a Revolve microscope (ECHO Laboratories Inc., San Diego, CA, USA). Cell viability percentage was calculated as the ratio between the number of viable cells and the number of total cells. Each cell proliferation assay was performed at least in three biological replicates.

MTT Assay

Cells were seeded in technical triplicates and according to the growth curve in 96-well plates at a density of 40,000–60,000 cells/well (in 50 μL complete medium phenol red-free), and subsequently, cells were exposed to drugs in an additional 50 μL medium. The external lanes and columns contained PBS only to avoid evaporation of the medium, and one lane was untreated control (no drug). Cultures were incubated at 37°C for 20 or 44 h after which cells were assayed with 10 μL of 5 mg/mL MTT reagent (Calbiochem, San Diego, CA, USA) in ultrapurified water for 4 h. Formazan crystals were dissolved overnight at 37°C with 100 μL of a solution containing 80% 2-propanol, 10% 1 N HCl, 10% Triton X-100. Metabolic activity was calculated as relative colorimetric changes measured at A570 (test) and A690 (reference) wavelengths, on a SpectraMax i3x plate reader (Molecular Devices, Sunnyvale, CA, USA). Calculated absorbance was normalized to untreated controls using Microsoft Excel software.

Flow Cytometry

Mitochondrial Transmembrane Potential Measurement

Detection of altered mitochondrial membrane potential was performed using the 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzim-idazolylcarbocyanine iodide (JC-1) MitoProbe detection kit (Thermo Fisher Scientific) and APC-conjugated Annexin V (BD Pharmingen) by flow cytometry. Cells were treated at increasing concentrations of MeSG up to 48 h, or cells were treated with 25 μM etoposide as positive controls. Cells were harvested and processed according to manufacturer’s protocol. The excitation wavelength was 488 nm, and emission fluorescent wavelengths were 575/25 nm (PE) and 530/30 nm (FITC) for JC-1, and the excitation wavelength was 635 nm, and the emission wavelength was 670/30 nm (APC) for Annexin V. Samples were processed with BD FACSCantoTM 10-Color (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Data were analyzed with BD FACSDiva software (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Annexin V positivity, as showed in the pictures of raw data (blue dots), was obtained via backgating process. Cell population distribution was graphed using Microsoft Excel software.

Cell Death by Apoptosis and Necrosis

Cells seeded in complete medium phenol red-free were treated accordingly. After the treatment, cells were resuspended in binding buffer (Abcam, Cambridge, MA, USA) at a concentration of 1 × 106 cells/mL. PE Annexin V and 7-aminoactinomycin D (5 μL/each; BD-Pharmingen, San Jose, CA, USA) were added to 100 μL of cell solution in binding buffer and incubated at room temperature for 15 min in the dark. After the incubation, 400 μL of binding buffer was added, and samples were analyzed with BD FACSCantoTM 10-Color (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) within 1 h. Cell death by apoptosis and necrosis was plotted in Prism (GraphPad Software, San Diego, CA, USA).

Cell Cycle Analysis in RPMI-8226 and H-929 Cell Lines

Cell cycle distribution was examined by measuring the cellular DNA content using flow cytometry. Cells were incubated for 24 h in RPMI-1640 phenol red- and FBS-free medium containing 150 ng/mL of nocodazole, then culturing back in RPMI-1640 phenol red-free medium with 10% FBS for 24 h. Cells were treated accordingly. After the treatment, 1 × 106 cells were collected, washed, and fixed with 70% cold ethanol. The cells were resuspended in 0.4 mL PBS solution containing RNase A and propidium iodide (Abcam, Cambridge, MA, USA) for 30 min at 37°C. Samples were analyzed with BD FACSCantoTM 10-Color (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Cell cycle distribution was analyzed by ModFit LTTM (VeritySoftware House, Topsham, ME, USA).

Cell Cycle Analysis in MM.1S Cell Line

This cell line markedly undergoes a process of endo-reduplication. In this case, cells were treated as above but collected in Krishan’s hypotonic detergent containing RNase A. Propidium iodide staining, samples, and cell cycle distribution modeling as above.

Immunoblotting

Treated cells were harvested in Laemmli buffer (2% SDS, 10% glycerol, 0.01% bromophenol blue, 62.5 mM Tris, pH 6.8, 0.1 M DTT), boiled at 95°C for 10 min, and centrifuged at 15,000g. Protein quantification was carried out by RC DC Protein Assay (BioRad, Hercules, CA, USA). Protein concentration was performed at 750-nm wavelength, on a SpectraMax plate reader (Molecular Devices, Sunnyvale, CA, USA). Data analysis was performed using Microsoft Excel software. Samples were boiled at 95°C for 10 min and centrifuged at 15,000g 30 min before loading into the gel wells. NuPAGETM 4–12%, Bis-Tris, Mini Protein Gels were run at 180 V in NuPAGETM MOPS (3-(N-morpholino)propanesulfonic acid) buffer (Thermo Fisher Scientific, Waltham, MA, USA) or 10% Mini-PROTEAN® TGXTM Precast (BioRad, Hercules, CA, USA). Gels were transferred onto polyvinylidene fluoride membrane at constant voltage (110 V/gel) for 2 h, at +4°C, in CAPS buffer, as previously described [18]. Post-transfer membranes were stained with Ponceau staining solution (Cell Signaling, Danvers, MA, USA) and gels in Coomassie Brilliant Blue R-250 (BioRad, Hercules, CA, USA) to confirm the efficiency of the transfer. Membrane was then blocked in 1X ScanLater Blocking Buffer (Molecular Devices, San Jose, CA, USA) for 1 h shaking at room temperature before incubating overnight at 4°C in primary antibody diluted in Tris-buffered saline +0.5% Tween 20 and 0.025% sodium azide. Primary antibodies (1:1,000 dilution; Cell Signaling, Danvers, MA, USA) phospho-AKT Ser473 (D9E), phospho-AKT Thr308 (#9275), pan-AKT (C67E7), caspase-8 (CASP-8) (1C12), caspase-9 (CASP-9) (C9), PARP-1 (46D11), phospho-Cdc25C Ser216 (63F9), Cdc25C (5H9), cyclin-B1 (D5C10), phospho-p53 Ser15 (16G8), p53 (DO-7), and p21Cip1/Waf1 (p21) p21(12D1) were used. Membranes were incubated at room temperature with secondary antibodies anti-mouse or anti-rabbit IgG HRP-linked (#7076 1:50,000 and #7074 1:5,000 dilution in blocking buffer, Cell Signaling, Danvers, MA, USA) for 45 min to 1 h before visualization. Bands were observed with the Western LightningTM ECL (PerkinElmer, Waltham, MA, USA) and CL-XPosureTM Film (Thermo Fisher Scientific, Waltham, MA, USA) or ChemiDoc (BioRad, Hercules, CA, USA) for AKT studies. Blot densitometry was evaluated with ImageJ software (Rasband, W.S., ImageJ, US National Institutes of Health, Bethesda, MD, USA, https://imagej.nih.gov/ij/, 1997–2018) and analyzed with Microsoft Excel software and graphed with Prism (GraphPad Software, San Diego, CA, USA). The protein of interest and the ratio between phosphorylated and pan protein values were normalized with the method of total protein normalization, as described in basic protocol 2 [20]. Results were plotted as relative intensity.

Statistical Analysis

GraphPad Prism version 8.0 was used to perform all statistical analyses. Two-way ANOVA followed by Sidak’s post hoc was applied to apoptotic mitochondria assay, and post hoc Dunnet’s test was used in all other experiments. The level of significance was established at p < 0.05. Assays were performed at least as two independent experiments. Data are presented as mean values ± SD, repeated at least in three biological replicates, in technical replicates when possible.

MeSG Reduced Cell Viability in MM Cells

To investigate the effect of MeSG on cell viability in MM cells, we used trypan blue and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays (Fig. 1a–f). Cell viability was determined up to 48 h using both assays. The viability decreased in a concentration- and time-dependent manner: cell viability was 50% with 50 nM of MeSG in RPMI-8226 after 24 h (Fig. 1a) and after 48 h in H-929 (Fig. 1b) and MM.1S (Fig. 1c) cell lines. To measure the activity of the oxidoreductase enzymes in the mitochondria, cells were seeded in 96-well plates with cell density according to the growth curve (data not shown). A decreased mitochondrial activity/viability was confirmed for all of 3 cell lines by using the MTT assay (Fig. 1d–f). Comparing the trypan blue assay with the inferred viability by MTT assay, there is a slight discrepancy in the results. In the trypan blue assay, data show that the cell lines H-929 and MM.1S reach values of about 50% viability with 50 nM of MeSG at 48 h (Fig. 1b, c); but the viability is lower than 50% in the same conditions by MTT assay (Fig. 1e, f). In RPMI-8226 cells, there is a discrepancy between the trypan blue assay (Fig. 1a) and the MTT assay (Fig. 1d). More specifically, with 5 nM treatment for 48 h, the viability by trypan is 60% versus MTT 6%; with 50 nM 24 h by trypan is 50% versus MTT 3%; and with 50 nM 48 h by trypan is 21% versus MTT 8%. Data also showed a different sensitivity to the compound by these cell lines. In fact, MTT activity decreased about 90% at 48 h with MeSG treatment of 5 nM in RPMI-8226 line (Fig. 1d) and of 500 nM in H-929 (Fig. 1e) and MM.1S lines (Fig. 1f). The EC80 for RPMI-8226 was 20 nM and for H-929 and MM. 1S was 270 nM, calculated by interpolation (data not shown). In aggregate, MeSG decreases cell viability and mitochondrial activity in a time- and concentration-dependent manner. The RPMI-8226 cell line is more sensitive to MeSG than the other 2 cell lines.

Fig. 1.

Cell viability and mitochondrial activity of human MM cell lines. (a, d) RPMI-8226, (b, e) H-929, and (c, f) MM.1S cell lines were incubated with increasing concentrations of MeSG at 24 and 48 h. The impact of the treatment was assessed (a–c) by trypan blue exclusion assay to measure cell viability and (d–f) MTT assay to measure mitochondrial activity. (a–c) Data are representative of three biological replicates (mean ± SD). (d–f) Data are presented as percentage of negative control (DMSO). Three biological replicates performed in technical triplicates (mean ± SD). Statistics: two-way ANOVA and Dunnett’s multiple comparisons by PRISM. *p< 0.05; **p< 0.01.

Fig. 1.

Cell viability and mitochondrial activity of human MM cell lines. (a, d) RPMI-8226, (b, e) H-929, and (c, f) MM.1S cell lines were incubated with increasing concentrations of MeSG at 24 and 48 h. The impact of the treatment was assessed (a–c) by trypan blue exclusion assay to measure cell viability and (d–f) MTT assay to measure mitochondrial activity. (a–c) Data are representative of three biological replicates (mean ± SD). (d–f) Data are presented as percentage of negative control (DMSO). Three biological replicates performed in technical triplicates (mean ± SD). Statistics: two-way ANOVA and Dunnett’s multiple comparisons by PRISM. *p< 0.05; **p< 0.01.

Close modal

MeSG Induces Loss of Mitochondrial Membrane Potential

To further investigate MeSG effect on mitochondria, changes in mitochondrial membrane potential (Δψm) were measured. To study the mitochondrial membrane potential (Δψm), we used JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimi-dazolylcarbocyanine iodide) staining. The cyanine dye JC-1 allows to discriminate between the functional statuses of mitochondria. Briefly, a collapse in Δψm from high potential (red fluorescent JC-1 aggregates) to lower potential (diffuse JC-1 green fluorescence) is a marker for early apoptotic event, simultaneously confirmed by the Annexin V staining. RPMI-8226, H-929, and MM.1S cells were treated with increasing concentrations of MeSG for 24 and 48 h, including a treatment with 25 µM etoposide as a positive control. To validate the assay, we treated controls cells with uncoupling agent carbonyl cyanide 3-chlorophenylhydrazone to induce Δψm loss. As expected, at increasing concentrations of MeSG, the cell line RPMI-8226 showed increased cell death since the increased positivity for Annexin V staining but unexpectedly not correlated to an increased mitochondrial loss of membrane potential over time (Fig. 2a). On the contrary, in the cell lines H-929 and MM.1S, the percentage of cells losing Δψm increased significantly and concurrently with increased percentage of apoptotic cells at increasing concentrations of MeSG (Fig. 2b, c). These results suggest that MeSG induces cell death in H-929 and MM.1S by processes that in part involve mitochondria depolarization. Cell death in RPMI-8226 is less associated with depolarized mitochondria. These data show for the first time that MeSG lowers mitochondria membrane potential, and this mechanism(s) need to be investigated in more detail.

Fig. 2.

Treatment with MeSG decreases mitochondrial membrane potential. (a) RPMI-8226, (b) H-929, and (c) MM.1S cell lines were incubated with increasing concentrations of MeSG up to 48 h. The effect of the treatment on mitochondrial membrane was assessed by JC-1 assay. A double staining with JC-1 and Annexin V was performed to measure apoptotic mitochondria. (a–c) Data are representative of three biological replicates (mean ± SD). DMSO is the negative control, and etoposide is the positive control. Statistics: two-way ANOVA and Šídák’s multiple comparisons by PRISM. *p< 0.05; **p< 0.01; ***p< 0.001; #p< 0.0001.

Fig. 2.

Treatment with MeSG decreases mitochondrial membrane potential. (a) RPMI-8226, (b) H-929, and (c) MM.1S cell lines were incubated with increasing concentrations of MeSG up to 48 h. The effect of the treatment on mitochondrial membrane was assessed by JC-1 assay. A double staining with JC-1 and Annexin V was performed to measure apoptotic mitochondria. (a–c) Data are representative of three biological replicates (mean ± SD). DMSO is the negative control, and etoposide is the positive control. Statistics: two-way ANOVA and Šídák’s multiple comparisons by PRISM. *p< 0.05; **p< 0.01; ***p< 0.001; #p< 0.0001.

Close modal

Treatment with MeSG Induces Apoptosis

To address apoptosis, RPMI-8226, H-929, and MM.1S cells were treated with increasing concentrations of MeSG up to 48 h and assessed by flow cytometry and Western blot assays (Fig. 3). For the apoptosis assays by flow cytometry, cells were stained with Annexin V and 7-aminoactinomycin D (Fig. 3a–c). The positive control for this assay was 25 μM etoposide. As in Figure 3a–f, all 3 cell lines showed a MeSG-induced increased percentage of cells in late apoptosis in a time- and concentration-dependent manner. More in detail, flow cytometry data confirmed the higher sensitivity of RPMI-8226 toward MeSG than H-929 and MM.1S cell lines. In fact, 20 nM of MeSG increased the percentage of apoptotic cells up to 60% at 24 h and 87% at 48 h in RPMI-8226 (Fig. 3a, d) but in the other 2 cell lines is below 60% up to 48 h for H-929 (Fig. 3b, e) and MM.1S (Fig. 3c, f). To obtain a cell population mostly apoptotic, it is required a treatment with MeSG increased of an order of magnitude (equal or higher than EC80 = 270 nM) (Fig. 3b, c, e, f). These results confirm that MeSG induces cell death, with cell population predominantly in late apoptosis for longer treatments at higher concentrations in all MM cell lines analyzed in this study. We also examined apoptotic markers in cell lysates (Fig. 3g–i, online suppl. S1; for all online suppl. material, see www.karger.com/doi/10.1159/000525299). For all of 3 cell lines, there is a strong trend in increased cleavage of caspase-8, especially for the fragment at 43 kDa (Fig. 3g–i, online suppl. S1). More in detail, for both cell lines H-929 (Fig. 3g, online suppl. S1b) and MM.1S (Fig. 3i, online suppl. S1c), high concentrations of MeSG at 48 h cause the activation of caspase-8. The treatment with MeSG caused an increased trend in caspase-9 cleavage and consequential activation but less marked (Fig. 3g–i, online suppl. S1). The cleavage of PARP-1, an important downstream effector of both intrinsic and extrinsic apoptotic pathways, is statistically relevant only in RPMI-8226 (Fig. 3g, online suppl. S1a) at high concentration of MeSG at 48 h. However, there is a trend in both the H-929 (Fig. 3h, online suppl. S1b) and MM.1S cell lines (Fig. 3i, online suppl. S1c). In aggregate, these data show that there is an increased activation of caspase-8 rather than caspase-9. Nevertheless, the activation of intracellular signaling seems to be a consequence of other mechanisms happening in the cell earlier upon treatment with MeSG.

Fig. 3.

Treatment with MeSG increases apoptosis and apoptotic markers caspase-8 and PARP-1 cleavage. Myeloma cells were incubated in the presence of increasing concentrations of MeSG. Cell apoptosis and necrosis were determined by flow cytometry with Annexin V and 7-AAD staining in (a, d) RPMI-8226; (b, e) H-929; and (c, f) MM.1S lines at 24 and 48 h. Markers for apoptosis were assessed by Western blot assay in (g) RPMI-8226, (h) H-929, and (i) MM.1S. Cells were treated with MeSG at cell-specific EC80 concentrations (20, 270, and 270 nM, respectively) up to 48 h. Etoposide, human Fas-ligand (=h-FAS-L), and staurosporine are controls. Data are representative of three biological replicates (mean ± SD). Statistics: one-way ANOVA and Dunnett’s multiple comparisons by PRISM. *p< 0.05; **p< 0.01, ***p< 0.001; #p< 0.0001.

Fig. 3.

Treatment with MeSG increases apoptosis and apoptotic markers caspase-8 and PARP-1 cleavage. Myeloma cells were incubated in the presence of increasing concentrations of MeSG. Cell apoptosis and necrosis were determined by flow cytometry with Annexin V and 7-AAD staining in (a, d) RPMI-8226; (b, e) H-929; and (c, f) MM.1S lines at 24 and 48 h. Markers for apoptosis were assessed by Western blot assay in (g) RPMI-8226, (h) H-929, and (i) MM.1S. Cells were treated with MeSG at cell-specific EC80 concentrations (20, 270, and 270 nM, respectively) up to 48 h. Etoposide, human Fas-ligand (=h-FAS-L), and staurosporine are controls. Data are representative of three biological replicates (mean ± SD). Statistics: one-way ANOVA and Dunnett’s multiple comparisons by PRISM. *p< 0.05; **p< 0.01, ***p< 0.001; #p< 0.0001.

Close modal

MeSG Triggers Cell Cycle Arrest at the G2 or M Phase of the Cell Cycle

Our data, so far, demonstrated that MeSG exerted a potent cytotoxic activity and had an inhibitory effect on cell line growth. To explore a possible mechanism leading to the loss of cells viability, cell cycle arrest was examined in MM cells. Cells were incubated with increasing concentrations of MeSG up to 48 h. A time- and concentration-dependent G2/M arrest was observed upon treatment with MeSG (Fig. 4). More in detail, RPMI-8226 showed the highest sensitivity to cell cycle arrest upon MeSG treatment (Fig. 4a, b), at the extent that the quantification of the cell cycle at 20 nM for 48 h was not possible because of the amount of debris (Fig. 4a, lower right panel, lilac curve). The cell line H-929 showed a slight sensitivity to the compound (Fig. 4c, d), and MM.1S showed sensitivity to the treatment at higher concentrations for longer treatment (Fig. 4e, f). Nocodazole treatment served as positive control for the arrest of cell cycle in G2/M [21]. In aggregate, these data showed that MeSG exerts a blockage of the cell cycle in G2/M phase, especially in RPMI-8226 cell line. We examined the effect of MeSG on the cell cycle markers, relevant for the progression from the late stage of G2 into mitosis (Fig. 5, online suppl. S2). The checkpoint known as G2/M targets cyclin-B1/CDK1 complex, which activation depends on Cdc25C. We interrogate the activity of Cdc25C and its phosphorylation status at Ser216 and cyclin-B1 protein levels. As shown in Figure 5 (online suppl. S2), MeSG does not increase the activation of Cdc25C but greatly decreases the levels of cyclin-B1 in all the MM cell lines. More in detail, the treatment with MeSG on the cell line RPMI-8226 decreases significantly the levels of cyclin-B1 at 48 h (Fig. 5a, online suppl. S2a). Because of the significant arrest in G2/M, which seems not determined directly by the activity of Cdc25C, we hypothesized a role of p21 as regulator of the late phases of the cell cycle. It is known that p21 may downregulate the complex cyclin-B1-CDK1, and p21 may be regulated by p53. Our data showed that p21 has an increasing trend especially at early time points (Fig. 5, online suppl. S2). More specifically, the cell line RPMI-8226, which showed the highest sensitivity to the induced-cycle arrest, showed a significantly increasing trend in p21 levels up to 24 h and a strong response to the positive control nocodazole (Fig. 5a, online suppl. S2a). However, active p53 levels were not increasing in any cell line, suggesting that the activation of p21 does not rely on p53 (Fig. 5, online suppl. S2). In aggregate, our data showed that MeSG causes a G2/M arrest likely supported by an increased trend in p21 levels in all cell lines, early on after the treatment.

Fig. 4.

Treatment with MeSG induces an arrest in the G2/M phase. (a, b) RPMI-8226, (c, d) H-929, and (e, f) MM.1S cells were treated with MeSG at increasing concentration for 24 and 48 h. Nocodazole is a positive control. Cell cycle arrest was determined by flow cytometry with PI-fixed staining after cell synchronization obtained with a pretreatment with nocodazole in FBS-free media. (a, c, e) Modeling of the cell cycle ModFit and (b, d, f) quantification. Representative of three biological replicates (mean ± SD). Statistics: two-way ANOVA and Dunnett’s multiple comparisons by PRISM. *p< 0.05; **p< 0.01; ***p< 0.001. PI, propidium iodide.

Fig. 4.

Treatment with MeSG induces an arrest in the G2/M phase. (a, b) RPMI-8226, (c, d) H-929, and (e, f) MM.1S cells were treated with MeSG at increasing concentration for 24 and 48 h. Nocodazole is a positive control. Cell cycle arrest was determined by flow cytometry with PI-fixed staining after cell synchronization obtained with a pretreatment with nocodazole in FBS-free media. (a, c, e) Modeling of the cell cycle ModFit and (b, d, f) quantification. Representative of three biological replicates (mean ± SD). Statistics: two-way ANOVA and Dunnett’s multiple comparisons by PRISM. *p< 0.05; **p< 0.01; ***p< 0.001. PI, propidium iodide.

Close modal
Fig. 5.

Treatment with MeSG decreases cyclin-B1 and increases p21 levels. (a) RPMI-8226, (b) H-929, and (c) MM.1S cells were treated with MeSG at cell-specific EC80 concentrations (20, 270, and 270 nM, respectively) up to 48 h. Insulin, H2O2 (hydrogen peroxide), nocodazole, and etoposide are controls. Cdc25C ratio (=cdc25 (Ser216)/cdc25), p53 ratio (= p53 [Ser15]/p53). Data are representative of three biological replicates (mean ± SD). Statistics: one-way ANOVA and Dunnett’s multiple comparisons by PRISM. *p< 0.05.

Fig. 5.

Treatment with MeSG decreases cyclin-B1 and increases p21 levels. (a) RPMI-8226, (b) H-929, and (c) MM.1S cells were treated with MeSG at cell-specific EC80 concentrations (20, 270, and 270 nM, respectively) up to 48 h. Insulin, H2O2 (hydrogen peroxide), nocodazole, and etoposide are controls. Cdc25C ratio (=cdc25 (Ser216)/cdc25), p53 ratio (= p53 [Ser15]/p53). Data are representative of three biological replicates (mean ± SD). Statistics: one-way ANOVA and Dunnett’s multiple comparisons by PRISM. *p< 0.05.

Close modal

MeSG Decreases AKT Activation

Bao et al. [16] have shown that schweinfurthins may decrease AKT activation in several cell lines. To dissect the triggering factor of the cytotoxicity exerted by MeSG on MM cell lines, we examined AKT activation (Fig. 6, online suppl. S3). The effect of MeSG on both AKT sites of phosphorylation was measured by Western blot analyses in cell lysates. The phosphorylation at both Ser473 and Thr308 sites was reduced in all the cells treated with MeSG up to 48 h. More in detail, the most sensitive cell lines for decreased phosphorylation at Ser473 site at earlier time points were H-929 (Fig. 6b, online suppl. S3b) and MM.1S (Fig. 6c, online suppl. S3c), compared to RPMI-8226 (Fig. 6a, online suppl. S3a). Time course analysis indicates that the treatment of RPMI-8226 with MeSG affected both sites of phosphorylation at longer exposure to the compound (Fig. 6a). The most sensitive cell line for decreased phosphorylation at Thr308 site is MM.1S (Fig. 6c), followed by RPMI-8226 (Fig. 6a) which showed a significant decreased only at 48 h. H-929 cell line showed a trend in decreased phosphorylation at Thr308 site only at 48 h (Fig. 6b). Taken together, these results indicate that MeSG is exerting a possible pleiotropic effect on a central signaling player, such as AKT, by decreasing its own phosphorylation status, thus decreasing its activation. The effect is most relevant and statistically significant at earlier time points in H-929 and MM.1S compared to RPMI-8226 for the Ser473 site and in MM.1S for the Thr308 site.

Fig. 6.

Treatment with MeSG decreases AKT activation (ratio p-AKT/AKT). (a) RPMI-8226, (b) H-929, and (c) MM.1S cells were treated with MeSG at cell-specific EC80 concentrations (20, 270, and 270 nM, respectively) up to 48 h. Insulin and wortmannin are controls. Data are representative of three biological replicates (mean ± SD). Statistics: two-way ANOVA and Dunnett’s multiple comparisons by PRISM. *p< 0.05; **p< 0.01.

Fig. 6.

Treatment with MeSG decreases AKT activation (ratio p-AKT/AKT). (a) RPMI-8226, (b) H-929, and (c) MM.1S cells were treated with MeSG at cell-specific EC80 concentrations (20, 270, and 270 nM, respectively) up to 48 h. Insulin and wortmannin are controls. Data are representative of three biological replicates (mean ± SD). Statistics: two-way ANOVA and Dunnett’s multiple comparisons by PRISM. *p< 0.05; **p< 0.01.

Close modal

Our studies of cell viability in myeloma cells showed an expected decrease in cell viability in a concentration- and time-dependent manner by trypan blue exclusion assay in all 3 cell lines. The viability, inferred by MTT assay, decreased as well in a concentration- and time-dependent fashion. Of note, the decrease in viability based on MTT assay readout appeared to be significantly different and more potent than that based on the trypan blue exclusion assay. Interestingly, we had noticed this discrepancy previously in the rat chondrosarcoma cell line LM1 [9]. In reviewing that study, we realized that the MTT assay would have indicated a greater cell GI than the inhibition with trypan blue, similarly to what we observed in myeloma cells.

The discrepancy in the viability assays led us to hypothesize that there could be a mitochondrial component to the decreased viability. To better understand the status of the mitochondria in all MM cell lines of this study, we investigated possible alteration in mitochondria membrane potential (Δψm). No previous studies have examined the role of mitochondria in triggering cell death upon treatment with schweinfurthin analogs. Our data show that the MeSG increased apoptotic mitochondria in H-929 and MM.1S but not RPMI-8226 cells. It is well known that MM cells produce extremely high levels of secreted proteins and rely on mitochondrial oxidative phosphorylation to produce energy to support it [22]. The mitochondria damage caused by MeSG led us to the hypothesis that MeSG is behaving in a similar fashion as some other well-known xenobiotics that increase the production of reactive oxygen species (ROS) [23, 24] or alter cholesterol homeostasis [24], leading to changes in mitochondria membrane permeability.

To better delineate the increased cell death caused by MeSG treatment, an apoptosis assay was performed by flow cytometry. The positivity for apoptotic cells, especially late apoptosis, increased in concentration- and time-dependent manner in all MM cell lines examined. Once again, RPMI-8226 is the most sensitive to MeSG treatment, followed by MM.1S and H-929. To discriminate what event(s) occur at early time points after treatments with MeSG, we investigated the key players of apoptosis, a specific mechanism of cell death, by Western blot. Previous studies have demonstrated increased executioner caspase 3 activity in LM1 chondrosarcoma cells with MeSG [9] and increased caspase-9 and PARP-1 cleavage in SF-295 cells [25]. We here looked at activation of caspase-9 and -8, as well as the downstream effector cleaved PARP-1. We aimed to confirm the involvement of the key players in the apoptotic event with an eye toward more firmly demonstrating either extrinsic (caspase-8) or intrinsic (caspase-9) apoptosis pathway involvement. Our data suggest that MeSG may activate caspase-8 rather than caspase-9. Nevertheless, in the absence of a strong effect on caspases and on PARP-1 at early time points, our data suggest apoptosis activation as a later event.

The literature has shown that an abnormal cell cycle is a common aspect in MM patients [26]. Currently, vincristine is an antimitotic drug used in the treatment of MM, which however has several collateral effects such as peripheral neuropathy [26]. This implies that cell cycle regulation is a therapeutic target for MM treatment, but antimitotic agents not targeting microtubules are needed. We have not yet investigated the microtubule integrity to assess possible side effects, but our novel compound MeSG showed a remarkable effect by triggering G2/M arrest. Currently, no data are available in the literature about the effect of MeSG on the cell cycle in MM cells. Flow cytometry data showed that MeSG induced cell cycle arrest in G2/M, with the highest sensitivity to the compound in RPMI-8226 cell line. To better dissect the key players involved in the arrest, we investigated the protein levels of markers involved in the regulation of the cell cycle. In case of G2/M arrest, a decrease in cyclin-B1 and a significant reduction in Cdc25C activation are expected. Our data showed that the decrease in cyclin-B1 is not dependent on significant reductions in Cdc25C activity. We instead showed in all cell lines examined that p21 may be the key regulator acting at early time points in favor of decreased cyclin-B1 levels. Additionally, this increased p21 activity is independent of p53. These new findings bring us to speculate if the cell cycle arrest may be caused by DNA damage and that at the best of our knowledge, has never been assessed for any of the schweinfurthin analogs before.

Another interesting aspect of this research is that the treatment with the analogue MeSG is decreasing the AKT signaling in all 3 cell lines. For a full activation, AKT is phosphorylated at the residues Ser273 and Thr308. Our findings demonstrate that MeSG strongly decreased AKT phosphorylation, therefore its activation, at both Ser273 and Thr308 sites in MM.1S and RPMI-8226 cell lines but decreasing only at 48 h in H-929 cells. Our data confirm the results obtained previously in melanoma cells, where the decreased AKT phosphorylation at Ser473 by 100 nM of schweinfurthin G has been linked to an arrest in G1 phase of the cell cycle [16]. In the last decade, the role of AKT has become more evident in the regulation of cell cycle, proliferation, and bioenergetics [27]. In the literature, selective AKT inhibitors alone or in combination have been extensively tested in MM cell lines, with promising results [13‒15]. Pan-AKT inhibitors alone (GSK690693) or in combination with other treatments are currently in clinical trials for treatment of lymphomas not responding to current therapies. However, the results in clinical trials, even though not always targeting MM disease alone, have revealed increased liver inflammation and risk of liver cancer and alteration of glucose homeostasis [15]. Novel compounds targeting the PI3K/AKT pathway and/or the downstream effectors are needed [28].

Changes in mitochondria membrane potential are in the same direction of AKT activation, where they decrease in MM.1S and H-929 and change less markedly in RPMI-8226. One hypothesis to support our observations is an early event happening in the cell that may change ROS homeostasis in the malignant plasma cells. It is known that ROS decrease phospho-AKT at Ser473 by altering the AKT heat-shock protein-90 complex which can protect phospho-AKT from dephosphorylation by protein phosphatase 2A [29, 30]. Also, ROS react with cholesterol-generating oxysterols in lipid rafts, leading to disruption of the rafts resulting in decreased AKT signaling and increased apoptosis [31]. ROS triggers a signal for FOXO genes to enter the nucleus to transcribe antioxidant genes, a process that is negatively regulated by AKT [32]. It is unknown if schweinfurthin treatment increases ROS in mammalian cells. In our previous work, we showed that schweinfurthins cause dramatic decreases in the cholesterol content of human glioblastoma and lung cancer cell lines [8, 33]. Cholesterol levels are tightly regulated and typically reduced levels will lead to activation of the sterol regulatory element-binding protein transcription factor which upregulates the synthesis of cholesterol [34]. Sterol regulatory element-binding proteins are activated in low cholesterol conditions by phosphorylation of AKT [35], so it is interesting that we see here a reduction in the AKT phosphorylation which should exacerbate the effects of the compounds on cholesterol homeostasis. Indeed, the 2 cell lines which show reduced AKT phosphorylation are less sensitive to the apoptotic inducing effects of the schweinfurthins, leading us to hypothesize this could be a mechanism of resistance to this effect. Further studies to unravel these hypotheses are now underway.

The authors would like to thank the flow cytometry core personnel at the Penn State Cancer Institute/College of Medicine Flow Cytometry Shared Resource. The compound TTI-3114 (MeSG) was supplied by IOThera Inc. (formerly Terpenoid Therapeutics Inc. by Material Transfer Agreement with Penn State College of Medicine).

No human or animal research was conducted in these studies. Ethical approval is not required for this study in accordance with local or national guidelines.

Jeffrey D. Neighbors and Raymond J. Hohl have financial and ownership interest in Terpenoid Therapeutics Incorporated and IOThera Incorporate which provided TTI-3114 (MeSG) for these experiments. Barbara Manfredi reports no conflicts of interest.

This work was funded by a Penn State University Professorship in Medical Oncology, the Miriam Beckner Cancer Research Endowment, and a gift from Highmark to the Penn State Cancer Institute.

Barbara Manfredi substantially contributed to the design of the work, data acquisition and analysis, as well as interpretation of data for the work, and drafted the work and revisited for critically important intellectual content. Jeffrey D. Neighbors and Raymond J. Hohl substantially contributed to the interpretation of data for the work and revisited for critically important intellectual content.

All data generated in this work is included in the figures of the manuscript. The data supporting the findings are kept at the Penn State University in the laboratory of the corresponding author and are available on request.

1.
Kazandjian
D
.
Multiple myeloma epidemiology and survival: a unique malignancy
.
Semin Oncol
.
2016
;
43
(
6
):
676
81
.
2.
Rajkumar
SV
,
Kumar
S
.
Multiple myeloma current treatment algorithms
.
Blood Cancer J
.
2020
;
10
(
9
):
94
.
3.
Janssens
R
,
Lang
T
,
Vallejo
A
,
Galinsky
J
,
Plate
A
,
Morgan
K
,
.
Patient preferences for multiple myeloma treatments: a multinational qualitative study
.
Front Med
.
2021 Jul
;
8
:
686165
.
4.
Naymagon
L
,
Abdul-Hay
M
.
Novel agents in the treatment of multiple myeloma: a review about the future
.
J Hematol Oncol
.
2016
;
9
(
1
):
52
.
5.
Neighbors
JD
,
Salnikova
MS
,
Beutler
JA
,
Wiemer
DF
.
Synthesis and structure-activity studies of schweinfurthin B analogs: evidence for the importance of a D-ring hydrogen bond donor in expression of differential cytotoxicity
.
Bioorg Med Chem
.
2006
;
14
(
6
):
1771
84
.
6.
Koubek
EJ
,
Weissenrieder
JS
,
Neighbors
JD
,
Hohl
RJ
.
Schweinfurthins: lipid modulators with promising anticancer activity
.
Lipids
.
2018
;
53
(
8
):
767
84
.
7.
Beutler
JA
,
Jato
JG
,
Cragg
GM
,
Wiemer
DF
,
Neighbors
JD
,
Salnikova
M
,
.
The schweinfurthins. Issues in development of a plant-derived anticancer lead
. In:
Medicinal and aromatc plants: agricultural, commercial, ecological, legl, pharmacological and social aspects
.
2006 May
. p.
301
9
.
8.
Holstein
SA
,
Kuder
CH
,
Tong
H
,
Hohl
RJ
.
Pleiotropic effects of a schweinfurthin on isoprenoid homeostasis
.
Lipids
.
2011
;
46
(
10
):
907
21
.
9.
Stevens
JW
,
Meyerholz
DK
,
Neighbors
JD
,
Morcuende
JA
.
5′-methylschweinfurthin G reduces chondrosarcoma tumor growth 
.
J Orthop Res
.
2018
;
36
(
4
):
1283
93
.
10.
Ho
ST
,
Lin
CC
,
Tung
YT
,
Wu
JH
.
Molecular mechanisms underlying yatein-induced cell-cycle arrest and microtubule destabilization in human lung adenocarcinoma cells
.
Cancers
.
2019
;
11
(
9
):
1384
.
11.
Castedo
M
,
Perfettini
JL
,
Roumier
T
,
Andreau
K
,
Medema
R
,
Kroemer
G
.
Cell death by mitotic catastrophe: a molecular definition
.
Oncogene
.
2004
;
23
(
16
):
2825
37
.
12.
Xiang
RF
,
Wang
Y
,
Zhang
N
,
Xu
WB
,
Cao
Y
,
Tong
J
,
.
MK2206 enhances the cytocidal effects of bufalin in multiple myeloma by inhibiting the AKT/mTOR pathway
.
Cell Death Dis
.
2017
;
8
(
5
):
e2776
10
.
13.
Ramakrishnan
V
,
Kimlinger
T
,
Haug
J
,
Painuly
U
,
Wellik
L
,
Halling
T
,
.
Anti-myeloma activity of Akt inhibition is linked to the activation status of PI3K/Akt and MEK/ERK pathway
.
PLoS One
.
2012
;
7
(
11
):
e50005
.
14.
Hirai
H
,
Sootome
H
,
Nakatsuru
Y
,
Miyama
K
,
Taguchi
S
,
Tsujioka
K
,
.
MK-2206, an allosteric akt inhibitor, enhances antitumor efficacy by standard chemotherapeutic agents or molecular targeted drugs in vitro and in vivo
.
Mol Cancer Ther
.
2010
;
9
(
7
):
1956
67
.
15.
Wang
Q
,
Chen
X
,
Hay
N
.
Akt as a target for cancer therapy: more is not always better (lessons from studies in mice)
.
Br J Cancer
.
2017
;
117
(
2
):
159
63
.
16.
Bao
X
,
Zheng
W
,
Hata Sugi
NH
,
Agarwala
KL
,
Xu
Q
,
Wang
Z
,
.
Small molecule schweinfurthins selectively inhibit cancer cell proliferation and mTOR/AKT signaling by interfering with trans-Golgi-network trafficking
.
Cancer Biol Ther
.
2015
;
16
(
4
):
589
601
.
17.
Mente
NR
,
Wiemer
AJ
,
Neighbors
JD
,
Beutler
JA
,
Hohl
RJ
,
Wiemer
DF
.
Total synthesis of (R,R,R)- and (S,S,S)-schweinfurthin F: differences of bioactivity in the enantiomeric series
.
Bioorg Med Chem Lett
.
2007
;
17
(
4
):
911
5
.
18.
Stern
KA
,
Place
TL
,
Lill
NL
.
EGF and amphiregulin differentially regulate Cbl recruitment to endosomes and EGF receptor fate
.
Biochem J
.
2008
;
410
(
3
):
585
94
.
19.
Sarin
V
,
Yu
K
,
Ferguson
ID
,
Gugliemini
O
,
Nix
MA
,
Hann
B
,
.
Evaluating the efficacy of multiple myeloma cell lines as models for patient tumors via transcriptomic correlation analysis
.
Leukemia
.
2020
;
34
(
10
):
2754
65
.
20.
Goldman
A
,
Harper
S
,
Speicher
DW
.
Detection of proteins on blot membranes: current protocols in protein science
.
Curr Protoc Protein Sci
.
2017
;
86
:
10.8.1
10.8.11
.
21.
Feng
R
,
Li
S
,
Lu
C
,
Andreas
C
,
Stolz
DB
,
Mapara
MY
,
.
Targeting the microtubular network as a new antimyeloma strategy
.
Mol Cancer Ther
.
2011
;
10
(
10
):
1886
96
.
22.
Rizzieri
D
,
Paul
B
,
Kang
Y
.
Metabolic alterations and the potential for targeting metabolic pathways in the treatment of multiple myeloma
.
J Cancer Metastasis Treat
.
2019
;
5
:
26
.
23.
Shankar
K
,
Mehendale
HM
.
Oxidative stress
.
Third Edit. Elsevier
;
2014
.
24.
Aisen
Y
,
Gatt
ME
,
Hertz
R
,
Smeir
E
,
Bar-Tana
J
.
Suppression of multiple myeloma by mitochondrial targeting
.
Sci Rep
.
2021
;
11
(
1
):
5862
10
.
25.
Kuder
CH
,
Sheehy
RM
,
Neighbors
JD
,
Wiemer
DF
,
Hohl
RJ
.
Functional evaluation of a fluorescent schweinfurthin: mechanism of cytotoxicity and intracellular quantification
.
Mol Pharmacol
.
2012
;
82
(
1
):
9
16
.
26.
Maes
A
,
Menu
E
,
Veirman
K
,
Maes
K
,
Vand Erkerken
K
,
De Bruyne
E
.
The therapeutic potential of cell cycle targeting in multiple myeloma
.
Oncotarget
.
2017
;
8
(
52
):
90501
20
.
27.
Keane
NA
,
Glavey
SV
,
Krawczyk
J
,
O’Dwyer
M
.
AKT as a therapeutic target in multiple myeloma
.
Expert Opin Ther Targets
.
2014
;
18
(
8
):
897
915
.
28.
Nitulescu
GM
,
Van De Venter
M
,
Nitulescu
G
,
Ungurianu
A
,
Juzenas
P
,
Peng
Q
,
.
The Akt pathway in oncology therapy and beyond (Review)
.
Int J Oncol
.
2018
;
53
(
6
):
2319
31
.
29.
Cao
J
,
Xu
D
,
Wang
D
,
Wu
R
,
Zhang
L
,
Zhu
H
,
.
ROS-driven Akt dephosphorylation at Ser-473 is involved in 4-HPR-mediated apoptosis in NB4 cells
.
Free Radic Biol Med
.
2009
;
47
(
5
):
536
47
.
30.
Wen
C
,
Wang
H
,
Wu
X
,
He
L
,
Zhou
Q
,
Wang
F
,
.
ROS-mediated inactivation of the PI3K/AKT pathway is involved in the antigastric cancer effects of thioredoxin reductase-1 inhibitor chaetocin
.
Cell Death Dis
.
2019
;
10
(
11
):
809
.
31.
Amiya
E
.
Interaction of hyperlipidemia and reactive oxygen species: insights from the lipid-raft platform
.
World J Cardiol
.
2016
;
8
(
12
):
689
.
32.
Zhao
Y
,
Hu
X
,
Liu
Y
,
Dong
S
,
Wen
Z
,
He
W
,
.
ROS signaling under metabolic stress: cross-talk between AMPK and AKT pathway
.
Mol Cancer
.
2017
;
16
(
1
):
79
12
.
33.
Inoshima
I
,
Inoshima
N
,
Wilke
G
,
Powers
M
,
Frank
K
,
Wang
Y
,
.
3-deoxyschweinfurthin B lowers cholesterol levels by decreasing synthesis and increasing export in cultured cancer cell lines
.
Lipids
.
2015
;
50
(
12
):
1195
207
.
34.
Horton
JD
,
Goldstein
JL
,
Brown
MS
.
SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver
.
J Clin Invest
.
2002
;
109
(
9
):
1125
31
.
35.
Jeon
TI
,
Osborne
TF
.
SREBPs: metabolic integrators in physiology and metabolism
.
Trends Endocrinol Metab
.
2012
;
23
(
2
):
65
72
.