Targeted nanocomplex carrying siRNA against MALAT1 sensitizes glioblastoma to temozolomide

Abstract

Intrinsic therapeutic resistance especially in cancer stem cells (CSCs) together with extensive tumor cell infiltration and restricted permeation of the blood-brain barrier (BBB) by drugs may all contribute to the treatment failure in patients with glioblastoma multiforme (GBM). Accumulating evidence suggests that long non-coding RNA (lncRNA), metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) plays a role in tumor cell infiltration and therapeutic resistance of GBM. Using our tumor-targeted nanocomplex, we have modulated the expression of MALAT1 and investigated its impact on GBM cells. Importantly, our nanocomplex is able to target CSCs that are considered to be the prime culprits in therapeutic resistance and recurrence of GBM. Attenuation of MALAT1 by RNA interference significantly lowered the growth, motility and stemness of GBM cells. In addition, silencing of MALAT1 clearly improved the sensitivity of GBM cells to chemotherapeutic agents including the current first-line therapy of GBM [temozolomide (TMZ)]. In animal models of GBM, tumor involution with a modest but statistically significant survival benefit was achieved with concurrent treatment of TMZ and nanocomplex-mediated silencing of MALAT1. These results suggest that combining standard TMZ treatment with lncRNA-targeting therapies using our nanocomplex could substantially enhance the very poor prognosis for GBM patients.

Figure 1.

Down-modulation of MALAT1 inhibits growth and migration of GBM cells. T98G cells were transfected with either empty targeted liposomes without payload (scL), scL encapsulating control siRNA (scL-siCTRL), siRNA targeting MALAT1 without the delivery system (Free siMAL), or scL encapsulating MALAT1 siRNA (scL-siMAL) at 100 nM siRNA concentration. (A, B) The MALAT1 levels were measured by quantitative RT-PCR 24 h after transfection. The assay was performed in triplicate and standard errors of the mean (SEM) was shown. (C, D) The effect of MALAT1 down-modulation on cell growth was monitored using CellTiter-Glo luminescence assay (C) and XTT assay (D). The experiments were performed in triplicate. (E) Cell cycle assays were performed in triplicate at 48 h after transfection. (F) Percent of apoptotic cells in sub-G1 phase. (G) Representative photographs of β-galactosidase staining 96 h after transfection. Assays were performed in triplicate. Scale bar, 100 μm. (H) Wound healing assay was performed in quadruplicate 24 h after transfection. Photomicrographs of the representative area 0 and 24 h after creating a wound. (I) Area of the scratch was quantified at 24 h. (J) Expression of proteins related to cell migration was determined 48 h after transfection using antibody array in duplicate. NS, not significant. *P < 0.001, **P < 0.05.

Figure 2.

MALAT1 in CSCs. Relative expression of CD133 (A, D), SSEA1 (B, E) and MALAT1 (C, F) was measured in 2D monolayer and 3D tumorsphere cultured T98G (A–C) or in magnetically sorted CD133⁻ and CD133⁺ T98G (D–F). Quantitative RT-PCR assay was performed in triplicate. GBM cells were transfected with scL-siMAL or control treatments at 100 nM siRNA concentration. (G) 48 h after transfection of U87R, expression of stem cell-related genes was monitored by flow cytometric analysis. (H) Induction of apoptosis was monitored at 48 h after transfection by Annexin V/PI staining in CD133⁻ and CD133⁺ population of T98G. Numbers in the quadrants indicate the percentage of cells in each quadrant. (I) Representative photographs of colony formation assays at 8 days after transfection of T98G. Colony formation assay was performed in sextuplicate. (J) Colony number in each treatment group was plotted. (K) Tumorsphere formation was assessed in sextuplicate at 10 days after transfection of T98G. *P < 0.001, **P < 0.05.

Figure 3.

Down-modulation of MALAT1 promotes in vitro sensitivity of GBM cells to TMZ. (A) Relative expression of MALAT1 in U87 and U87R (TMZ-resistant clone derived from U87). Quantitative RT-PCR assay was performed in triplicate. (B) The correlation between MALAT1 levels and TMZ IC₅₀ values in human GBM cell lines. All values measured in triplicate and the average is shown. R² = 0.670. TMZ-resistant T98G cells were treated with 1000 μM TMZ for 72 h with or without receiving pre-treatment with 100 nM scL-siMAL for 24 h. The experiment was performed in triplicate. (C) Photomicrographs of a representative area. (D) Percent of apoptotic cells in sub-G1 phase. (E) Flow cytometric analyses of Annexin V/7-AAD double-staining. (F) CASP3 antibody staining in CD133⁻ and CD133⁺ population. (G) XTT assays 48 h after treatment with various concentrations of TMZ. Assay was performed in triplicate. *P < 0.05, **P < 0.001.

Figure 4.

Down-modulation of MALAT1 inhibits anti-apoptotic genes. T98G cells were transfected with 100 nM scL-siMAL for 24 h, followed by 1000 μM TMZ for 72 h. The controls included treatments with either scL, scL-siCTRL, or free siMAL in combination with TMZ as well as with TMZ alone. Expression of MALAT1 (A), MGMT (B), and MRP1 (C) were assessed by RT-PCR performed in triplicate. (D) Expression of anti-apoptotic proteins was determined using antibody array performed in duplicate. NS, not significant. *P < 0.05, **P < 0.001.

Figure 5.

Down-modulation of MALAT1 promotes chemosensitivity of GBM cells. T98G (A–C) and U87R (D–F) cells were transfected with either scL-siMAL or scL-siCTRL at 100 nM for 24 h, followed by treatment with BCNU (A, D), cisplatin (B, E), and irinotecan (C, F) at various concentration. XTT assay was performed in triplicate 72 h after treatment with chemotherapeutic drugs and IC₅₀ values determined.

Figure 6.

MALAT1 interacts with p53 tumor suppressor gene. (A) U87R was transfected with either scL-siCTRL or scL-siMAL at 100 nM for 24 h. Expression of MALAT1 and p53 were measured by the TaqMan assay. (B) U87R cells were transfected with scL encapsulating p53 plasmid DNA (scL-p53) at a concentration of 7 μg DNA/dish for 24 h. Expression of p53 and MALAT1 were measured by the TaqMan assay in triplicate. (C) The correlation between p53 and MALAT1 levels in intracranial T98G tumor either treated with scL-siMAL or untreated. Each data point indicates individual tumor (8 to 10 tumors per group). (D) Average expression of p53 and MALAT1 in intracranial T98G tumor from (C). (E) T98G cells were transfected with either 50 nM scL-siMAL or 50 ng scL-p53 as a single agent or in combination of both for 24 h followed by TMZ treatment. XTT assay was performed in triplicate 72 h after treatment with various concentrations of TMZ. *P < 0.001, **P < 0.05.

Figure 7.

MALAT1 down-modulation inhibits GBM tumor growth. Mice bearing intracranial U87-luc2 tumors were treated with scL-siMAL and/or TMZ as shown in (A). N = 5 mice per group. (B) Bioluminescence images (BLI) of the intracranial U87-luc2 tumor are shown from before (day 10) and after the treatments (day 21). Bioluminescence signals, shown in a color map, correlate with tumor sizes. Red color: stronger signal, Violet color: weaker signal. (C) Change in bioluminescence intensity of individual tumor was quantitated. (D) Bioluminescence intensities of tumors were plotted as a function of time. (E) Brain tumors were weighed at harvest on day 23. N = 4 to 5 tumors per group *P < 0.05, **P < 0.001.

Figure 8.

Increased apoptosis in TMZ-resistant GBM tumors and mouse survival after treatment with a combination of scL-siMAL plus TMZ. Mice bearing intracranial T98G tumor were treated with scL-siMAL and/or TMZ as shown in (A). N = 10 to 11 mice per group. After receiving two injections of each treatment (on day 16), five mice from each group were euthanized and brain tumors were harvested and weighed (B). Single cells were isolated from tumors and stained for CASP3 and PARP antibodies. Representative histograms are shown in (C). CASP3⁺ (D) and PARP⁺ (E) cells were quantified to assess apoptosis in tumors. (F) Kaplan–Meier survival curves of mice (log-rank P = 0.029). *P < 0.05, **P < 0.001.