Phospho-valproic acid (MDC-1112) suppresses glioblastoma growth in preclinical models through the inhibition of STAT3 phosphorylation

Abstract

New therapeutic strategies against glioblastoma multiforme (GBM) are urgently needed. Signal transducer and activator of transcription 3 (STAT3), constitutively active in many GBM tumors, plays a major role in GBM tumor growth and represents a potential therapeutic target. We have documented previously that phospho-valproic acid (MDC-1112), which inhibits STAT3 activation, possesses strong anticancer properties in multiple cancer types. In this study, we explored the anticancer efficacy of MDC-1112 in preclinical models of GBM, and evaluated its mode of action. MDC-1112 inhibited the growth of multiple human GBM cell lines in a concentration- and time-dependent manner. Normal human astrocytes were resistant to MDC-1112, indicating selectivity. In vivo, MDC-1112 reduced the growth of subcutaneous GBM xenografts in mice by up to 78.2% (P < 0.01), compared with the controls. Moreover, MDC-1112 extended survival in an intracranial xenograft model. Although all vehicle-treated mice died by 19 days of treatment, 7 of 11 MDC-1112-treated mice were alive and healthy by the end of 5 weeks, with many showing tumor regression. Mechanistically, MDC-1112 inhibited STAT3 phosphorylation at the serine 727 residue, but not at tyrosine 705, in vitro and in vivo. STAT3 overexpression rescued GBM cells from the cell growth inhibition by MDC-1112. In addition, MDC-1112 reduced STAT3 levels in the mitochondria and enhanced mitochondrial levels of reactive oxygen species, which triggered apoptosis. In conclusion, MDC-1112 displays strong efficacy in preclinical models of GBM, with the serine 727 residue of STAT3 being its key molecular target. MDC-1112 merits further evaluation as a drug candidate for GBM. New therapeutic options are needed for glioblastoma. The novel agent MDC-1112 is an effective anticancer agent in multiple animal models of glioblastoma, and its mechanism of action involves the inhibition of STAT3 phosphorylation, primarily at its Serine 727 residue.

Figure 1.

MDC-1112 inhibits GBM cell growth. (A) Chemical structure of MDC-1112 (Phospho-valproic acid). (B) IC₅₀ values for glioblastoma cells treated with MDC-1112 or VPA for 24 h. These values are representative of three experiments, each performed in triplicates; results were within 10%. The table shows the 13.4- to 25.4-fold enhancements in potency of MDC-1112 over VPA in four human GBM cell lines. (C) MDC-1112 reduces human U87 cell growth in a concentration- and time-dependent manner. Results are expressed as % control. (D) MDC-1112 inhibits GBM cell colony formation in a concentration-dependent manner in U87 and LN-18 cells. (*P < 0.05, versus control). (E) Differential cytotoxic effect of MDC-1112 in GBM cells compared with the NHA. MDC-1112 inhibits human GBM cancer cell growth in a concentration-dependent manner. Cell growth was determined in U118, LN-229 and LN-18 GBM cells and in NHA after treatment with escalating concentrations of MDC-1112 for 48 h. Results are expressed as % control. *Significantly different compared with all other cell lines (P < 0.05, one-way analysis of variance test).

Figure 2.

MDC-1112 reduces GBM xenograft growth. (A) MDC-1112 inhibits the growth of human U87 xenografts. U87 cells were injected s.c. into the flank areas of nude mice, and when palpable tumors were observed, the mice received MDC-1112 (50 mg/kg per day) in PBS or just PBS (control) by intraperitoneal injection for 15 days. U87 tumor volume growth over time for vehicle control- and MDC-1112-treated mice. *Significantly different compared with control group [P < 0.01, one-way analysis of variance (ANOVA) test]. (B) Mice weight progression for control and MDC-1112-treated mice. (C) MDC-1112 inhibits the growth of human U118 xenografts. U118 cells were injected s.c. into the flank areas of nude mice, and when palpable tumors were observed, the mice received MDC-1112 (50 mg/kg per day) in PBS or just PBS (control) by intraperitoneal injection for 30 days. U118 tumor volume growth over time for vehicle control- and MDC-1112-treated mice. *Significantly different compared with control group (P < 0.01, one-way ANOVA test). (D) Tumor weight at sacrifice. *Significantly different compared with control group (P < 0.01, one-way ANOVA test). (E) Body weight over time of mice bearing U118 xenografts treated with vehicle control (PBS) or MDC-1112 50 mg/kg. All values: mean ± SD. (F) Ki-67 and p21 immunostaining were performed on U87 tumor sections and photographs were taken at ×20 magnification. Representative images are shown. The consecutive section was stained with isotype IgG as negative staining control and it is shown in the upper right corner. Quantification is displayed on the right. Results were expressed as percent of Ki-67⁺ or p21⁺ cells ± SEM per ×20 field. *Significant compared with control group; P < 0.05. (G) Ki-67 and cleaved caspase 3 (CC3) immunostaining were performed on U118 tumor sections and photographs were taken at ×20 magnification. Representative images are shown. The consecutive section was stained with isotype IgG as negative staining control and it is shown in the upper right corner. Quantification is displayed on the right. Results were expressed as percent of Ki-67⁺ cells ± SEM per ×20 field. *Significant compared with control group; P < 0.05.

Figure 3.

Treatment with MDC-1112 extends survival in mice bearing GBM intracranial tumors. (A) U87-Luc cells were intracranially injected, and when tumor size was confirmed by bioluminensence, mice received MDC-1112 (50 mg/kg 5×/week) in PBS or just PBS (control) by intraperitoneal injection for up to 35 days. Tumor growth over time until endpoint in vehicle control (red) or MDC-1112 (blue)-treated mice determined by bioluminescent imaging. (B) MDC-1112 therapy significantly prolonged survival of animals as compared with vehicle control group (P < 0.005). Kaplan–Meier survival curve of vehicle control (red) or MDC-1112-treated mice (blue) is shown. Seven of eleven MDC-1112-treated animals were intentionally killed (censored) for histological analysis of brain at day 35 despite being healthy. (C) Tumor growth was monitored by bioluminescent imaging in mice bearing intracranial U87-Luc cells (n = 10–11 per group). Representative IVIS images of U87-Luc-bearing animals over time treated as indicated.

Figure 4.

MDC-1112 inhibits STAT3 Ser727 signaling in vitro and in vivo. (A) Immunoblots of STAT3, phosphorylated STAT3 at the Ser727 residue (p-STAT3^Ser727), from U87 cells treated with MDC-1112, 1 × IC₅₀ for different periods of time. Bands were quantified and results are shown as the ratio p-STAT3^Ser727:STAT3. Values are mean ± SEM. *P < 0.05 versus control. (B) Immunoblots of STAT3 and phosphorylated STAT3 at the Ser727 residue (p-STAT3^Ser727), from U87 and LN-18 cells treated with various concentrations of MDC-1112 for 4 h. Bands were quantified and results are shown as the ratio p-STAT3^Ser727:STAT3. Values are mean ± SEM. *P < 0.05 versus control. (C) Immunostaining for phosphorylated STAT3 at the Ser727 residue (p-STAT3^Ser727) or Tyr705 residue (p-STAT3^Tyr705) or p-ERK1/2 expression on tissue sections of U87 tumors from control and MDC-1112-treated mice (×20). Representative images are shown. The consecutive section was stained with isotype IgG as negative staining control and it is shown in the upper right corner. Quantification is displayed on the right. Results were expressed as percent of p-STAT3^Ser727, p-STAT3^Tyr705 or p-ERK1/2 positive cells per field. *Significant compared with control group; P < 0.05. (D) Immunoblots of ERK1/2 and phosphorylated ERK1/2 (p-ERK), from U87 cells treated with various concentrations of MDC-1112 for 4 h. Bands were quantified and results are shown as the ratio p-ERK:ERK. Values are mean ± SEM. *P < 0.05 versus control. (E) Immunoblots of STAT3, p-STAT3^Ser727, ERK1/2 and phosphorylated ERK1/2 (p-ERK), from U87 tumor lysates. Loading control: β-actin. Each lane represents a different tumor sample. Bands were quantified and results are expressed as the ratio of phospho over total expression levels for each protein. Values are mean±SEM. *P < 0.05 versus control. (F) STAT3 overexpression ameliorates, in part, the cell growth inhibition by MDC-1112. U118 cells were transfected with a control (cDNA) or STAT3-expressing plasmid for 48 h and then treated with 50 or 100 µM MDC-1112 for 48 h. Cell growth was evaluated by the MTT assay; *P < 0.05 versus control. Top: STAT3 expression status in whole cell protein lysates following transfection. (G) Effect of silencing STAT3 on MDC-1112-induced cell growth reduction. U118 cells were transfected with either control or STAT3 small interfering RNA. After transfection, cells were treated with MDC-1112 for 24 h and cell growth was evaluated; *P < 0.05 versus control. Immunoblots to verify STAT3 silencing were performed on whole cell extracts obtained from these cells (top panel).

Figure 5.

MDC-1112 reduces mitochondrial STAT3 levels and induces mitochondrial ROS in GBM cells. (A) Immunoblots for STAT3, Hsp60 or COX IV in mitochondrial (MF) fractions from U87 cells treated with MDC-1112 for 3 h. Bands were quantified and results expressed as percent control for each protein. Values are mean ± SEM. *P < 0.05 versus control. (B) Immunoblots for STAT3, phosphorylated STAT3 at the Ser727 residue (p-STAT3^Ser727), GRIM-19 and COX IV in MF from U118 and LN-18 cells treated with MDC-1112 for 3 h. Bands were quantified and results expressed as percent control for each protein. Values are mean ± SEM. *P < 0.05 versus control. (C) Immunoblots of GRIM-19 in total fractions from U87 cells treated with various concentrations of MDC-1112 for 4 h. Bands were quantified and results are shown as the ratio GRIM-19:β-actin. (D) U87 and LN-18 cells were treated with MDC-1112 for 1 h as indicated. The levels of superoxide anion in the mitochondria were determined by flow cytometry using the MitoSOX-Red fluorescent probe. Bar graph analysis of Geometric means of cells that underwent MitoSOX Red Staining. (E) U87 and LN-18 cells were treated without (Control) or with MDC-1112 1 × IC₅₀ or VPA (1 × IC₅₀) for 1 h. Superoxide anion levels in the mitochondria was determined by confocal microscopy. Representative images are shown. (F) Treatment with Mito-TEMPO prevents the increase in MitoSOX-Red induced by MDC-1112 in various GBM cells. Values are mean ± SEM. *P < 0.05 versus control.

Figure 6.

MDC-1112 induces intrinsic apoptosis in GBM cells. (A) MDC-1112 collapses the mitochondrial membrane potential (ΔΨm) in a concentration-dependent manner. Cells were stained with JC-1 and analyzed with flow cytometry after treatment with MDC-1112 for 3 h. Data were quantified and results are shown as mean ± SEM; *P < 0.05 versus control. (B) Immunoblots for full-length caspase 9 in total cell protein extracts from LN-18 or U87 cells treated with MDC-1112, as indicated, for 24 h. Loading control: β-actin. Bands were quantified and results are shown as the ratio cleaved:β-actin; *P < 0.05 versus control. (C) Immunoblots for full length and cleaved caspase 3 in total cell protein extracts from LN-18 or U118 cells treated with MDC-1112, as indicated, for 24 h. Loading control: β-actin. Bands were quantified and results are shown as the ratio cleaved:full length protein; *P < 0.05 versus control. (D) Immunoblots for full length and cleaved PARP in total cell protein extracts from LN-18 or U87 cells treated with MDC-1112, as indicated, for 24 h. Bands were quantified and results are shown as the ratio cleaved/full length protein; *P < 0.05 versus control. (E) Cell death by apoptosis was determined by flow cytometry using the dual staining (Annexin V and PI) in U87 cells treated with increasing concentrations of MDC-1112 for 24 h. Results are expressed as fold-increase compared with the percentage of Annexin V (+) cells in the control group. (F) Cell death by apoptosis was determined by flow cytometry in LN-18 and LN-229 cells incubated without or with MDC-1112 1 × IC₅₀ for 24 h. Results are expressed as fold-increase compared with the percentage of apoptotic cells in the control group.