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. 2025 Sep 25;15:32869. doi: 10.1038/s41598-025-14731-4

A novel HPβCD-Cu(DDC)2 delivery system in patient derived orthotopic xenograft targeting MGMT-mediated temozolomide resistance in glioblastoma

Hamid Suhail 1, Md Ataur Rahman 2, Mahesh Kumar Yadab 2, Sunalee Gonawala 1, Ana deCarvalho 1, James R Ewing 1, James Snyder 1, Meser M Ali 1,2,✉
PMCID: PMC12464313  PMID: 40998915

Abstract

The uniform lethality of glioblastoma (GBM) with a survival of less than 2 years despite best available therapy is attributed to treatment resistance due to DNA repair mechanisms that drive disease relapse and tumor heterogeneity. One prognostic factor identified as a reliable biomarker for GBM sensitivity to temozolomide (TMZ) and radiotherapy (RT) is the overexpression of O6-methylguanine-methyl-transferase (MGMT) enzyme. Patients with active MGMT were found to receive little benefit from TMZ and RT. They represent a group of great unmet need with no treatment options that significantly improve survival. Recently, several preclinical and clinical studies suggest that the alcohol aversion drug, disulfiram (DSF), inhibited MGMT and improved the efficacy of TMZ in GBM when combined with copper (Cu). However, phase II trial showed that there was no significant survival benefit from oral Cu/DSF. Nevertheless, the major limitation of oral Cu/DSF has been delivery of fragile DSF to the in vivo system. To address this limitation, we developed a novel delivery system using 2-hydroxypropyl beta cyclodextrin (HPβCD) encapsulating the Cu complex of DSF’s active metabolite, diethyldithiocarbamic acid (DDC). It was determined that HPβCD stabilized Cu(DDC)2. In vitro cell culture study revealed that HPβCD-Cu(DDC)2 inhibited MGMT through the ubiquitin-proteasome pathway. Inhibition of MGMT activity in cell cultures vastly increased the alkylation-induced DNA double-strand breaks, cytotoxicity, and the levels of apoptotic markers like histone family member X (γ-H2AX), JNK-P and cleavage of Poly [ADP-ribose] polymerase 1 (PARP-1). Preliminary intravenous delivery of HPβCD-Cu(DDC)2 in combination with TMZ in an MGMT-positive patient derived orthotopic xenograft (PDOX) model demonstrated tumor size regression. HPβCD-Cu(DDC)2 targets MGMT-145-cysteine and its unique cytotoxic mechanism circumvents MGMT-mediated TMZ resistance. This novel delivery system shows promise for overcoming MGMT-mediated resistance in GBM, offering a potential new therapeutic strategy.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-14731-4.

Keywords: Glioblastoma (GBM), O6-methylguanine-methyltransferase (MGMT), Temozolomide (TMZ), Disulfiram (DSF), 2-Hydroxypropyl beta cyclodextrin (HPβCD), Ubiquitin-proteasome pathway, Brain tumor

Subject terms: Cell signalling, Pharmacology, Biochemistry, Cell biology, Drug discovery, Molecular biology, Oncology

Introduction

Annually, around 38,000 Americans are diagonized with primary malingnant brain tumors1,2. Glioblastoma (GBM), designated as a World Health Organization (WHO) category 4 neoplasm, is the most prevalent and malignant primary brain tumor in adults3,4. GBM is a malignant glioma characterized by molecular heterogeneity and the poorest prognosis. Despite the advances made in therapeutic options for GBM, its prognosis remains poor with an overall survival rate (< 2 years) that has been stagnant for three decades at approximately 20%. It is one of the unfortunate cancers where no predominant genetic alteration has been identified that could be targeted, resulting in limited therapeutic options5. The multimodal treatment of GBM includes maximal surgical resection followed by radiotherapy (RT) plus temozolomide (TMZ) chemotherapy-increases median survival to 12–15 months, although the disease typically progresses within 6–9 months, and 2-year survival is less than 25% 6.

In clinical practice, approximately 60% of GBM patients have shown overexpression of MGMT, an enzyme responsible for DNA repair. These patients were found to receive little benefit from treatment by TMZ and RT8–10 ; however, TMZ remains the standard primary therapy for nearly all GBM patients due to the lack of effective alternatives. GBM patients with high MGMT expression had significantly shorter survival when compared to patients with low expression of MGMT8. Since overexpression of MGMT is correlated with resistance to chemotherapy and RT, therefore, inhibition of MGMT is considered an attractive therapeutic target for GBM11. However, MGMT inhibitors are yet to be introduced into routine clinical practices. Therefore, much attention has been paid to pharmacological inhibitors of MGMT. Chemical depletion using O6-benzylguanine derivatives as MGMT inhibitors have been actively tested, but these compounds are associated with high hematologic toxicity12,13. Thus, MGMT inhibitors are yet to be introduced into routine clinical practices.

Recently, disulfiram (DSF), an FDA-approved medication for alcoholism, has attracted interest as a possible supplementary therapy in GBM owing to its capacity to suppress MGMT14. DSF, in conjunction with copper (Cu), generates the complex Cu(DDC)2, which amplifies the DNA-damaging effects of TMZ and other alkylating drugs such as carmustine (BCNU)15. Preclinical studies indicate that Cu(DDC)₂ promotes MGMT degradation through the ubiquitin-proteasome pathway, enhancing the therapeutic efficacy of TMZ and RT against GBM16,17. Evidence indicates that Cys145, a reactive cysteine residue in MGMT, is essential for DNA repair and functions as the principal binding site for diethyldithiocarbamate (DDC) (Fig. 1)18. The clinical use of DSF has been limited by its fast degradation into DDC after oral treatment19. The decomposition occurs in the stomach’s acidic milieu and persists in the bloodstream, where enzymes such as glutathione (GSH) further decompose DDC20. The amount of DDC reaching the brain is markedly reduced, hence limiting its effectiveness in treating GBM. It is, therefore, crucial that DDC is properly delivered by vehicles that are not only reliable and effective in overcoming cellular and physiological barriers. A possible strategy involves utilizing 2-hydroxypropyl beta cyclodextrin (HPβCD), a cyclic oligosaccharide capable of encapsulating hydrophobic pharmaceuticals such as HET0016, hence enhancing their stability, solubility, and bioavailability21,22. HPβCD has been extensively used for its capacity to traverse biological barriers, including the blood-brain barrier, rendering it an option for the delivery of Cu(DDC)2 to GBM and other malignant brain tumors23,24. In the present study, we present an HPβCD-Cu(DDC)2 delivery method designed to enhance TMZ efficacy by targeting MGMT-mediated resistance in GBM. This method seeks to improve therapeutic efficacy by overcoming MGMT-mediated resistance in GBM patients.

Fig. 1.

Fig. 1

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Synthesis of Cu(DDC)2 with hydroxypropyl-β-cyclodextrin. (A) DSF is metabolized into diethyldithiocarbamate (DDC), a potent copper chelator, which forms a Cu(DDC)2 complex. This copper-binding complex plays a critical role in DSF’s anti-tumor activity. (B) Cu(DDC)2interacts with HPβCD in aqueous solution at 25 °C to form the (Cu(DDC)2–HPβCD) inclusion complex. HPβCD encapsulates the Cu(DDC)2complex within its hydrophobic cavity, enhancing its solubility, stability, and bioavailability for potential therapeutic applications.

Materials and methods

Chemicals

Cell culture media was from Thermo Scientific (Waltham, MA), and fetal bovine serum was purchased from Hyclone (Logan, Utah). Cell culture grade DMSO was purchased from Fischer Scientific (PA). Anhydrous hydroxypropyl-β-cyclodextrin (HPβCD was purchased from Sigma-Aldrich (St. Louis, MO) and used without further purification. Diethyldithiocarbamate was purchased from Tokyo Chemical Industry Co, Ltd, Tokyo, Japan. The UV-vis spectrum analyses were conducted with an Agilent Technologies Cary 60 instrument. The concentration of Cu(DDC)2 was determined using a linear equation derived from a standard calibration curve of Cu(DDC)2 in DMSO. The molecular weight of the inclusion complex was analyzed using MALDI-TOF spectrometry at Scripps Research Institute, California.

Synthesis of Cu(DDC)2 with HPβCD

Cu(DDC)2 was synthesized according to literature methods as described elsewhere25. The product complex was highly insoluble in water as shown in Fig. 2. In the present study, we optimized Cu(DDC)2 formulation with 2-Hydroxypropyl Beta Cyclodextrin (HPβCD). HPβCD is a derivative of β-cyclodextrin that has been extensively used as a drug delivery vehicle and recently a treatment for Niemann Pick Type C disease26,27. Moreover, due to its small size (~ 1 nm) of the HPβCD encaged drug, bio-elimination of the drug proceeds through the kidneys. We have synthesized HPβCD-Cu(DDC)2 complex by following our previous method28 as shown in Fig. 1.

Fig. 2.

Fig. 2

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Absorption spectra measurement of HPβCD-Cu(DDC)2. (A) Absorption spectra of HPβCD-Cu(DDC)2 (red), DDC (green), HPβCD (pink) and Cu(DDC)2 (brown) in water. Only HPβCD-Cu(DDC)2 (red) complex absorbs light at 437 nm. (B) Absorption spectra of HPβCD-Cu-(DDC)2 with added GSH (850 µM) in H2O at pH 7.4 for 8 h. (C) Absorption spectra of Cu-(DDC)2 with added 850 µM of GSH in 10% DMSO at pH 7.4 for 8 h. Encapsulation of Cu-(DDC)2 inside HPβCD provided stability against glutathione (GSH).

Briefly, Cu(DDC)2 was dissolved in DMSO and then was added slowly to a 10% HPβCD solution in sterile water with continuous vortex. Then, the resulting yellow brown solution was diluted with distilled water and was freeze dried on lyophilizer to obtain yellowish powder. The final complex, HPβCD-Cu(DDC)2 was dissolved in water and purified by dialysis (MW cutoff size > 1,000 g). We have characterized HPβCD-Cu(DDC)2 by UV-visible and MALDI-TOF (Supplementary Figure S1). MALDI-TOF of the product showed average molecular masses 1777.89 and 1563.73 for [HPβCD-Cu(DDC)2+K]+ and [HPβCD-DDC+K]+, respectively. The HPβCD-Cu(DDC)2 complex is highly soluble in water while Cu(DDC)2 is completely insoluble in water as shown in Figure 2. The absorbance spectra of HPβCD-Cu(DDC)2, Cu(DDC)2, DCC, and HPβCD (in H2O) are shown in Figure 2. Only HPβCD-Cu(DDC)2 complex showed an absorption maxima (λ max) at 437 nm originating form Cu(DDC)2 transition metal complex. Therefore, encapsulation of Cu(DDC)2 with HPβCD did not shift the absorption maxima of free Cu(DDC)2. Free Cu(DDC)2 (in DMSO) was used to generate a standard curve for the estimation of Cu(DDC)2 content in HPβCD-Cu(DDC)2 complex (Supplementary Figure 2). Therefore, the amount of Cu(DDC)2 encaged inside HPβCD was determined by spectroscopic method. The drug loading efficiency (LE) and encapsulation efficiency (EE) was calculated 6.5% and 30.3%, respectively, using follow formula:

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Tumor cells

Patient-derived GBM cells (HF2303) were characterized at Henry Ford Hospital following previously established protocols29,30. We obtained this HF2303 cells from Dr. deCarvalho’s lab at Henry Ford Hospital and the cells were grown in neurosphere medium (NM), composed of DMEM/F-12 supplemented with N2 (Gibco), 0.5 mg/ml BSA (Sigma), 25 µg/ml gentamicin (Gibco), 0.5% antibiotic/antimycotic (Invitrogen), 20 ng/ml basic fibroblast growth factor, and 20 ng/ml EGF (Peprotech).

Cell viability MTT assay

An MTT experiment was used to assess cellular vitality in HF2303 cells. HF2303 cells were inoculated onto 96-well plates at a density of 1 × 104 cells per well. The cells underwent treatment with different concentrations of TMZ, HPβCD-Cu(DDC)2, Cu(DDC)2, HPβCD, and DDC, or no treatment control for a duration of 72 h. After the treatment time, 10 µL of MTT reagent (5 mg/mL in PBS) was introduced to each well, and the plates were incubated at 37 °C for 2–4 hours to facilitate the production of formazan crystals. The medium was meticulously aspirated, and 100 µL of DMSO was introduced to each well to solubilize the formazan crystals. Absorbance was quantified at 570 nm utilizing a microplate reader. Cell viability of treated cells was quantified as a percentage relative to untreated control cells. The data represent the average of three independent experiments (n = 3), expressed as Mean ± Standard Error (SE), with findings shown as bar graphs to depict the dose-dependent effects of each treatment on HF2303 cell viability.

In vivo orthotopic xengraft

Female nude rats (RNU nu/nu) weighing 150–170 g obtained from Charles River Laboratory (Frederick, MD) were used in these experiments. HF2303 cells (200k in 5µL), were implanted orthotopically at 3 mm to the right and 1 mm anterior to bregma according to published methods31,32.

In vivo MRI imaging.

MRI studies were performed in a Varian/Magnex (Santa Clara, California), 7 Tesla, 20 cm bore magnet system with a Bruker console using Paravision 6.0 software. Gradient maximum strengths and rise times were 250 mT/m and 120 µs respectively. All MRI image sets were acquired with a 32 × 32 mm field of view. Transmit and receive coils were a Bruker Quadrature Birdcage (transmit) and a 4-channel phased-array surface coil receiver (Rapid MR International, Columbus OH). High-resolution T1-weighted images were acquired pre- and post-administration of the DOTArem (GdDOTA, 0.1 mmolGd/kg) contrast agent with the following parameters: matrix: 256 × 192, 27 slices, 0.5 mm thickness, no gap, NE = 1, NA = 4, TE/TR = 16/800 ms.

Tumor volume quantification

Tumor volumes were calculated by manually delineating the hyperintense tumor region on each coronal T1-weighted image slice using ImageJ (NIH, Bethesda, MD, USA) by following our previous reports22,33,34. The area of the tumor in each slice was measured, and the total tumor volume was calculated by summing the tumor areas across all slices and multiplying by the slice thickness (0.5 mm). Tumor volumes were expressed as mean ± SEM and compared among groups using one-way ANOVA followed by Tukey’s post hoc test. A p-value < 0.05 was considered statistically significant.

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Hematoxylin-eosin (H&E) staining

Whole brain tumor tissues were collected from rats administered TMZ and HPβCD-Cu(DDC)₂ for histological examination. The tissues were formalin fixed (in 10% neutral-buffered formalin for 24 h) and paraffin embedded (FFPE). Serial sections with a thickness of 5 μm were produced using a microtome and affixed to glass slides. The sections were deparaffinized using xylene, rehydrated through a graded ethanol series, and washed with distilled water. For hematoxylin-eosin (H&E) staining, the slides were initially treated with Harris hematoxylin for 5 min, thereafter, rinsed under running tap water to eliminate surplus stains. Sections were distinguished using 1% acid alcohol and subsequently blued in Scott’s tap water replacement. The slides were subsequently counterstained with eosin for 2 min, then dehydrated in escalating doses of ethanol and cleared in xylene. Coverslips were affixed with a xylene-based mounting medium. Photographs of the stained slices were obtained with a light microscope at different magnifications. Photographs of tumor tissues from animals administered TMZ in conjunction with HPβCD-Cu(DDC)₂ were captured at 40X magnification. Control animals were observed, and all perished due to tumor load by the 16th week, preventing further analysis in that group.

Immunohistochemistry (IHC)

Tumor specimens from the control, TMZ-treated, and TMZ + HPβCD-Cu(DDC)₂-treated groups were collected at the end of the trial. The tumors were precisely removed, briefly washed in phosphate-buffered saline (PBS), and promptly fixed in 10% neutral-buffered formalin for 24 h at ambient temperature. Tissues were subjected to fixation, followed by processing through a graded sequence of alcohol, clearing in xylene, and embedding in paraffin wax blocks. FFPE tumor blocks were sliced to a thickness of 5 μm with a microtome and affixed on glass slides. The slides were deparaffinized in xylene for 10 min and rehydrated through a sequential alcohol series (100%, 95%, and 70%) for 5 min each, followed by a rise in distilled water. The stained sections were examined using a light microscope (specify type if known) at multiple magnifications (e.g., 10×, 20×, 40×). The morphology of tumors and histological alterations due to treatment were assessed by examining cell density, nuclear structure, and indications of necrosis or apoptosis. Photomicrographs were obtained for documentation and subsequent study.

Ki-67 immunohistochemical staining

Whole brain tumor tissue sections were collected from rats administered TMZ and HPβCD-Cu(DDC)₂ for histological examination. The sections were fixed in 10% formalin, embedded in paraffin, and cut into 6-µm thick slices. After deparaffinization and rehydration, antigen retrieval was performed using a citrate buffer (pH 6.0) heated to 95 °C for 20 min. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 min at room temperature. The sections were then incubated overnight at 4 °C with a primary antibody against Ki-67 (dilution: 1:200). After washing with PBS, a secondary antibody conjugated with horseradish peroxidase (HRP) was applied for 1 h at room temperature. Immunoreactivity was visualized using a diaminobenzidine (DAB) chromogen substrate. Finally, the slides were dehydrated, cleared, and mounted. Ki-67 positive cells were quantified using ImageJ software. The percentage of Ki-67-positive cells was determined by analyzing multiple fields per section with ImageJ, and statistical comparisons were performed to assess Prism program 10.

Western blot analysis

After treatment, GBM cells were harvested and lysed in cell lysis buffer supplemented with protease inhibitors. Protein concentrations were quantified using the Bradford method. An equal amount of denatured protein samples was separated by SDS-PAGE electrophoresis and then transferred onto a PVDF membrane. PVDF membranes were blocked with 5% non-fat milk containing 0.1% Tween-20 for 1 h at room temperature and then incubated with primary antibody at 4 °C overnight. After washing with TBST three times, the membranes were incubated with second antibody at room temperature for ~ 1 h. The expression of target proteins was detected by electro chemiluminescence (ECL) using protein bands were detected. The intensities of the proteins were determined by densitometric scanning and analyzed by ImageJ software.

Ethical approval of animal experiment

All procedures performed in the study involving animals were in accordance with the ethical standards in accordance with the ARRIVE guidelines of Henry Ford Health (Approval number: 1467). The experimental design, methodology, and analysis adhere to the principles outlined to ensure transparency and reproducibility in animal research.

Ethical statement

The animal experiments were performed according to the NIH guidelines and the experimental protocol was approved by the Institutional Animal Care and Use Committee of Henry Ford Health (Approval number: 1467). Body weight was measured twice weekly as an indicator of overall animal health. Euthanasia for moribund animals was performed in a CO2 chamber.

Statistical analysis

Results are indicated as mean ± SEM. Statistical analysis was examined by one-way analysis of variance followed by contrast analysis using Prism program 10 (GraphPad Software Inc., San Diego, CA, USA). The significance of the experiments was calculated by a Student t test. All the values of p < 0.05 were considered significant.

Results

Disulfiram (DSF) is the active ingredient in the drug Antabuse used for many years in aversion therapy for chronic alcoholism. DSF also has the potential to be used as a treatment for neoplastic diseases35. Phenotypically, DSF induces apoptosis36inhibits cell proliferation37and reduces angiogenesis by reducing invasion and metastasis38,39. Although the anti-cancer properties of DSF have been known for over 50 years40very little is known about the activity of this drug on brain tumors. DSF is easily metabolized as diethyldithiocarbamate (DDC), a strong Cu chelator, Cu(DDC)2 as shown in Fig. 1.

In vitro stability of HPβCD-Cu(DDC)2

In order to probe the stability of HPβCD-Cu(DDC)2 under the physiological condition that HPβCD-Cu(DDC)2 encounter during circulation after IV administration, we monitored absorption spectra of Cu(DDC)2 and HPβCD-Cu-(DDC)2 with added 850 µM of glutathione enzyme (GSH) for 8 h (Fig. 2). It has been reported that the concentration of GSH in extracellular fluid is about 10 µM41while intracellular GSH is significantly higher, in the range of 1–11 mM42,43. The intensity of absorption maxima at 437 nm originating from Cu(DDC)2 decreased over the period of time (Fig. 2). After 8 h, the absorption intensity at 437 nm for Cu(DDC)2 almost decreased near the baseline (Fig. 2C) indicating the decomposition of Cu(DDC)2 in solution. In contrast, we did not observe any spectral change at 437 nm for HPβCD-Cu(DDC)2 over the period of time (Fig. 2B). Therefore, HPβCD-Cu(DDC)2 is highly stable under physiological conditions. HPβCD-Cu(DDC)2 is stable both at 4˚C and room temperature for weeks without any precipitation of Cu(DDC)2. While the current oral formulation (Cu/DSF) that is given with naked Cu faces a serious challenge for decomposition and neurotoxicity44HPβCD-Cu(DDC)2 complex provided stability and safety for in vivo applications.

Evaluation of cytotoxicity of HPβCD-Cu(DDC)₂ against MGMT-positive (HF2303) and MGMT-negative (HF3253) cells

To evaluate the cytotoxic effects of HPβCD-Cu(DDC)2 and its each component against MGMT-positive HF2303 cells, an MTT assay was performed. TMZ treatment (50–200 µM) had a minimal effect on cell viability; even at 200 µM, cell viability remained high (~ 80–90%), indicating strong TMZ resistance. HPβCD-Cu(DDC)₂ (25–200 nM) exhibited dose-dependent cytotoxicity, significantly reducing viability. Free Cu(DDC)₂ (25–200 nM) also reduced viability, but to a lesser extent than the HPβCD-formulated version. HPβCD alone and DDC alone showed no significant cytotoxicity, indicating the observed effects are specific to the Cu(DDC)₂ formulation (Fig. 3A). However, HF3253 (MGMT-negative cells), TMZ treatment showed higher cytotoxicity compared to MGMT-positive cells, indicating TMZ sensitivity due to lack of MGMT expression. Cu(DDC)₂ complex also showed cytotoxic effect but was less potent than that of HPβCD-Cu(DDC)₂. As in HF2303 cells, HPβCD and DDC alone did not show any cytotoxic effect (Fig. 3B).

Fig. 3.

Fig. 3

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Effect of TMZ on cellular viability of both MGMT-positive (HF2303) and MGMT-negative (HF3253) glioma cell lines. (A, B) Bar graphs displaying the dose-dependent effects on cell viability in HF2303 and HF3253 cells after 72 h of treatment with TMZ, HPβCD-Cu(DDC)2, Cu(DDC)2, HPβCD, and DDC. Results are expressed as mean ± SEM and are representative of three independent experiments. Significant differences between control and treated with different concentration is indicated (ns = nonsignificant; *P < 0.05, **P < 0.01, ***P < 0.001). (C) Western blot analysis was conducted to evaluate the expression levels of MGMT protein in both MGMT-positive (HF2303) and MGMT-negative (HF3253) cell lines. (D) The intensities of the proteins were determined by densitometric scanning and analyzed by ImageJ software, with expression levels were normalized to GAPDH. To ensure accurate and reliable comparisons, the expression levels of the target proteins were normalized against GAPDH.

The Western blot demonstrates MGMT protein expression in the two patient-derived GBM cell lines, HF2303 (MGMT-positive) shows a strong MGMT band and HF3253 (MGMT-negative) exhibits no detectable MGMT band (Fig. 3C). HF2303 cells show a high MGMT expression while HF3253 cells have a negligible MGMT expression (Fig. 3D). HPβCD-Cu(DDC)₂ exhibits strong cytotoxic effects in both MGMT-positive and MGMT-negative GBM cells, with significantly enhanced potency in MGMT-upregulated HF2303 cells compared to TMZ. This suggests HPβCD-Cu(DDC)₂ can overcome TMZ resistance mediated by MGMT expression. HPβCD-Cu(DDC)₂ formulation clearly improves drug delivery and bioactivity over the free Cu(DDC)₂ compound. These results support further investigation of HPβCD-Cu(DDC)₂ as a novel and effective therapeutic strategy for TMZ-resistant GBM.

Inhibition of MGMT by HPβCD-Cu(DDC)₂ in HF2303 cells promotes DNA damage and apoptosis

The inhibitory effects of HPβCD-Cu(DDC)₂ against MGMT overexpressing patient derived cells, HF2303 were investigated using western blot analysis. As in Fig. 4 A, treatment with HPβCD-Cu(DDC)₂ (5 µM) in combination with TMZ (100 µM) for 24 h significantly reduced MGMT levels. The depletion of MGMT in tumor cells is attributed to its degradation via the ubiquitin-proteasome pathway. Importantly, the 24-hour inhibition of MGMT function led to a pronounced increase in alkylation-induced DNA double-strand breaks, as evidenced by elevated levels of γ-H2AX (Fig. 4). This was accompanied by heightened cytotoxicity and the activation of apoptotic pathways, indicated by increased phosphorylation of JNK (phosphor-JNK) and the cleavage of PARP-1 (Fig. 4). These findings suggest that HPβCD-Cu(DDC)₂ treatment potentiated the cytotoxic effect of TMZ enhances as evidenced by DNA damage response which triggers apoptosis in HF2303 cells, making it a potential strategy to sensitize HF2303 to alkylating agents like TMZ. The addition of the proteasome inhibitor MG-132 led to a significant accumulation of MGMT in the HF2303 cells, as shown in Fig. 5. This finding suggests that the degradation of MGMT in these cells occurs through the ubiquitin-proteasome pathway. By inhibiting the proteasome, MG-132 prevents the normal breakdown of MGMT, leading to its accumulation within the cells. Therefore, this indicates that MGMT is targeted for proteasomal degradation, and its depletion from tumor cells is mediated through the ubiquitin-proteasome system.

Fig. 4.

Fig. 4

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Effect of HPβCD-Cu(DDC)₂ in combination with TMZ on MGMT in HF2303 cells. (A) HF2303 cells were treated with HPβCD-Cu(DDC)₂ and TMZ (100 µM), leading to the inhibition of MGMT. Represented western blot resulted in the upregulation of key molecular markers associated with DNA damage response and apoptosis, including γ-H2AX (a marker of DNA double-strand breaks), poly (ADP-ribose) polymerase-1 (PARP-1), and c-Jun N-terminal kinase (JNK). (B) The intensities of the proteins were determined by densitometric scanning and analyzed by ImageJ software, with expression levels were normalized to β-actin. Results are expressed as mean ± SE and representatives of three independent experiments (n = 3, ns = nonsignificant; statistical test: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Fig. 5.

Fig. 5

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Proteasome Inhibitor MG-132 Induces Accumulation of MGMT in HF2303 Tumor Cells. (A) HF2303 cells were treated with MG-132 at a concentration of 50 µM. Following treatment, Western blot analysis was performed to assess the levels of MGMT protein. (B) The intensities of the proteins were determined by densitometric scanning and analyzed by ImageJ software, with expression levels were normalized to β-actin. Results are expressed as mean ± SE and representatives of three independent experiments (n = 3, statistical test, ***P < 0.001, ****P < 0.0001).

MGMT-Cys145 as the target for HPβCD-Cu(DDC)2

The human MGMT protein contains five cysteine residues, with Cys145 being the most reactive and responsible for accepting alkyl groups during DNA repair45. Studies have shown that Cys145 is the only site of conjugation by disulfiram (DSF) in the MGMT protein, making it critical for its function46. To investigate whether HPβCD-Cu(DDC)2 reacts with Cys145, a biotin-labeled, sulfhydryl-reactive HPDP probe (Kayman Inc.), which mimics the SH-BG probe known to bind specifically to cysteine residues, was used47. Purified recombinant MGMT protein (GenScript) was incubated with SH-HPBP-biotin and dithiothreitol (DTT) either before or after treatment with HPβCD-Cu(DDC)2. Following this, SDS-PAGE was performed, and protein-bound biotin was detected using streptavidin–horseradish peroxidase on the blots. The rMGMT proteins were purified by dialysis (with a molecular weight cutoff of > 3,000 Da) to remove DTT, HPDP-biotin, and any residual drug prior to loading on SDS-PAGE. Results showed that when the protein was exposed to HPβCD-Cu(DDC)2 and DTT, followed by SH-HPDP-biotin treatment, there was a notable reduction in probe binding (Fig. 6, lane 3), suggesting that HPβCD-Cu(DDC)2 effectively blocked the active site cysteine (Cys145) in the human rMGMT protein. Additionally, reprobing the blot with MGMT antibody confirmed that the rMGMT protein was present at consistent levels across all assays (Fig. 6, lower panel).

Fig. 6.

Fig. 6

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HPβCD-Cu(DDC)₂ Interaction Effectively Blocks the Active Site Cysteine (Cys145) in Human rMGMT Protein. (A) Wild-type rMGMT (C145-Cys) protein was incubated with 5 µM HPβCD-Cu(DDC)₂ and 5 mM DTT for 20 min. HPDP-biotin (5 µM) was then added, and the incubation continued for 15 min. The samples were subjected to electrophoresis, transferred to a membrane, and probed with streptavidin–horseradish peroxidase to detect protein-bound biotin (upper panel). The blot was subsequently reprobed with antibodies against MGMT (lower panel). (B) The intensities of the proteins rMGMT and MGMT analyzed by ImageJ software and the expression levels were normalized to MGMT. Results are expressed as mean ± SE and representatives of three independent experiments (n = 3, **P < 0.01).

Treatment of HF2303 (MGMT+) tumor with HPβCD-Cu(DDC)2

In a separate group of 4 rats implanted with MGMT-positive patient-derived cells (HF2303), 4 nude rats with intracerebral HF2303 tumors were assessed 16 weeks post-implantation. Tumor localization was performed using MRI at week 10, followed by the administration of a therapeutic regimen of HPβCD-Cu(DDC)2 (56 mg/kg based on Cu(DDC)2), given intravenously five days per week for 4 weeks. TMZ was given orally for 4 weeks at a dose of 50 mg/kg (five days per week). Magnetic Resonance Imaging (MRI) showed a marked reduction in tumor size in the group receiving the combined treatment compared to the groups treated with TMZ alone. Tumors in the combination group showed significantly smaller volumes at the endpoint indicating effective therapeutic response. The tumor mass in the combination-treated group was substantially smaller, with less infiltration into surrounding brain tissues, compared to control or TMZ-treatment groups. This reduction suggests that combined therapy effectively suppresses tumor progression and limits invasive growth (Fig. 7A). Therefore, the combined treatment with HPβCD-Cu(DDC)₂ and TMZ significantly reduced tumor size in PDOX model of GBM with upregulated MGMT (Fig. 7B). Thus, the brain drug delivery system shows promise as an effective approach to overcome TMZ resistance and therapeutic benefit in MGMT-positive PDOX tumor model. Histopathological evaluation using Hematoxylin-Eosin (H&E) staining highlighted pronounced morphological differences between treatment groups. In HPβCD-Cu(DDC)₂ and TMZ combination-treated tumors, there was a clear reduction in cellular density, with increased necrotic regions, indicating higher tumor cell death (Fig. 7C). Conversely, untreated or TMZ-treated tumors displayed dense, actively proliferating tumor cells and limited necrosis when analysis with 40X (Fig. 7D). Additionally, Ki-67 immunohistochemical staining revealed a significant reduction in proliferative tumor cells in rats treated with TMZ + HPβCD-Cu(DDC)₂ compared to control and TMZ alone (Fig. 7E). These data suggest that HPβCD-Cu(DDC)₂ synergizes with TMZ to effectively suppress tumor cell proliferation (Fig. 7F). Therefore, it has been demonstrated that the combined treatment with HPβCD-Cu(DDC)₂ and TMZ significantly reduced tumor size in a patient derived orthotopic xenograft (PDOX) model of GBM.

Fig. 7.

Fig. 7

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Efficacy of HPβCD-Cu(DDC)₂ in overcoming TMZ resistance in MGMT-positive HF2303 tumor models. (A) Post-contrast T1-weighted MR images of HF2303 tumor 16-weeks post-injection with Gd-DOTA. (B) Tumor volume measurements in GBM-bearing animals following treatment with TMZ alone or in combination with HPβCD-Cu(DDC)₂. Data are presented as mean ± SEM (***P < 0.001). (C) Hematoxylin-eosin (H&E) staining of whole brain tumor tissue from animals treated with TMZ and HPβCD-Cu(DDC)₂. (D) 40X image of H&E staining of tumor tissue from animals treated with TMZ combined with HPβCD-Cu(DDC)₂. (E) Ki-67 immunohistochemical staining of whole brain tumor sections from rats treated with TMZ and HPβCD-Cu(DDC)₂. (F) Ki-67-positive cells were quantified using ImageJ software. Percentage of Ki-67-positive cells in tissue sections slide from rats treated with control, TMZ, and TMZ + HPβCD-Cu(DDC)₂ were analyzed using prism software. Results are expressed as mean ± SE and representatives of three independent slide (n = 3, *P < 0.05, ***P < 0.001).

HPβCD-Cu(DDC)2 inhibits MGMT expression in TMZ-treated glioma PODXs

After MRI images, the animals (n = 4) were sacrificed and tumors were collected. The collected frozen tumors and contralateral brains were cut into small pieces, and proteins were extracted for western blot analysis, following methods outlined in previous publications48,49. HF2303 (T1 and T2) tumors showed upregulated MGMT expression after treatment with TMZ (Fig. 8A). However, tumors treated with a combination of HPβCD-Cu(DDC)2 and TMZ (T3 and T4), MGMT expression was depleted. Notably, the contralateral brains from both TMZ and TMZ + HPβCD-Cu(DDC)2 treated groups (B1, B2, B3, and B4) did not exhibit any MGMT expression (Fig. 8A). Additionally, immunohistochemistry (IHC) analysis of MGM protein expression was also evaluated in glioma PDOXs to assess the effect of the treatments. In TMZ-treated PDOXs, a modest reduction in MGMT staining intensity and the number of MGMT-positive cells was observed, correlating with partial tumor reduction. In the combination treatment group HPβCD-Cu(DDC)₂+TMZ, IHC analysis revealed a dramatic decrease in MGMT expression. Staining intensity was significantly reduced, and MGMT-positive cells were sparse in tumor sections (Fig. 8B). Therefore, these results suggest that combination of HPβCD-Cu(DDC)₂ and TMZ induced significant tumor reduction in PDOXs, largely through the potent inhibition of MGMT, highlighting its potential as a brain-penetrating drug delivery system for effective GBM therapy.

Fig. 8.

Fig. 8

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HPβCD-Cu(DDC)2 potently inhibits MGMT expression in TMZ-treated glioma PDOXs. (A) Expression of MGMT in the representative HF2303 cases at the contralateral brain tissues (B1, B2) and tumor tissues (T1, T2) from TMZ treated brains. Similarly B3, B4 (contralateral brains) and T3, T4 (tumors) from TMZ + HPβCD-Cu(DDC)2 treated brains. (B) Immunohistochemical (IHC) staining for non-degraded MGMT in TMZ-treated tumors. IHC staining showing MGMT degradation in TMZ + HPβCD-Cu(DDC)₂-treated tumors.

Possible mechanism of HPβCD-Cu(DDC)₂ in overcoming TMZ resistance in GBM

MGMT restores guanine to its normal state by transferring a methyl group from O6-methylguanine (O6-MeG) to its active site cysteine residue (Cys-145)50. Once MGMT receives the methyl group, it becomes inactivated and is subsequently degraded via ubiquitin-mediated pathways12. We hypothesize a similar “suicidal” enzyme reaction occurs when the thiol group of MGMT-Cys-145 reacts with DDC, resulting in an S-S bond formation between MGMT-Cys-145 and DDC, as illustrated in Fig. 9. Upon entering into GBM cancer cells, DDC dissociates from the Cu(DDC)₂ chelate due to the presence of high intracellular concentration of glutathione, which is about a thousand times higher than in normal cells51.

Fig. 9.

Fig. 9

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Possible mechanism of MGMT inactivation and degradation. TMZ induces methylation of DNA and overexpression of MGMT reverses this methylation, resulting in TMZ resistance and promoting tumor growth and cell survival. The combination of HPβCD-Cu(DDC)₂ with TMZ leads to oxidative modification of the active-site cysteine residue (Cys-145) of MGMT, converting it into an inactive disulfide form. This inactivated MGMT is subsequently targeted for degradation via the ubiquitin-proteasome pathway. Loss of MGMT activity leads to unrepaired DNA double-strand breaks, activation of DNA damage response markers such as γ-H2AX and PARP, and ultimately triggers apoptotic cell death. This mechanism offers a strategy to overcome TMZ resistance in glioblastoma therapy.

Discussion

This study introduces a novel therapeutic strategy to overcome MGMT-mediated resistance in GBM using a new delivery system, HPβCD-Cu(DDC)₂. Despite advances in surgery, radiotherapy, and TMZ, the prognosis for GBM patients remains poor, particularly for those with elevated MGMT expression. MGMT plays a crucial role in DNA repair by removing alkyl groups from the O6 position of guanine, diminishing the efficacy of alkylating agents like TMZ52. Recent reports by other investigators, support the development of targeted delivery systems to inhibit MGMT and enhance GBM therapy53. Thus, we compare our findings with related studies, discuss implications for clinical practice, and highlight the relevance of our results across the treatment landscape.

Several studies have explored DSF and Cu as adjunct therapies to overcome MGMT-mediated resistance in GBM14,54. For instance, it has been demonstrated that DSF/Cu complexes induce proteasomal degradation of MGMT, thereby increasing tumor sensitivity to TMZ and other alkylating agents55. However, that work highlighted the challenges associated with the rapid degradation of DSF in the bloodstream and limited brain penetration, limiting its therapeutic effect. Our current study addresses these issues by encapsulating Cu(DDC)₂ (DSF’s active metabolite) in HPβCD to enhance stability, solubility, and targeted delivery to GBM cells across the blood-brain-barrier (BBB). In contrast to traditional DSF/Cu oral formulations, our intravenous HPβCD-Cu(DDC)₂ complex remains highly stable in circulation, even in the presence of physiological concentrations of GSH (Fig. 2). This is a key improvement over prior studies where the DSF/Cu complex degraded before inducing any significant anti-tumor effect. Additionally, our results shows that enhancing drug stability improves the therapeutic outcome in brain tumors by facilitating BBB penetration, which is critical for effective glioma therapy56. Our recent study demonstrated that HET0016-HPβCD formulation induced anti-angiogenic effect in HF2303 PDOX model22.

Our study showed that HPβCD-Cu(DDC)₂ effectively inhibits MGMT through the ubiquitin-proteasome pathway, resulting in increased DNA damage, marked by elevated γ-H2AX levels (Fig. 4). This is consistent with the findings demonstrated that proteasomal inhibition leads to sensitization of GBM cells to alkylating agents through the accumulation of DNA lesions57. Notably, our work further confirmed the degradation of MGMT at the reactive cysteine residue (Cys145), a finding that supports earlier research on the role of disulfiram in modifying MGMT function at this critical site46. Inhibition of MGMT not only enhances the cytotoxic effects of TMZ but also triggers apoptotic pathways, evidenced by increased PARP-1 cleavage (Fig. 4). In line with our results, Stein et al. reported that proteasome-mediated degradation of MGMT enhances therapeutic efficacy and promotes apoptosis when combined with alkylating agents57.

HPβCD-Cu(DDC)₂ addresses a critical barrier in GBM treatment by inhibiting MGMT-mediated DNA repair mechanism58 to potentiate TMZ efficacy in patients with upregulated MGMT expression, who typically respond poorly to standard of care therapy. The stability of HPβCD-Cu(DDC)₂ in physiological conditions indicates that this delivery system can overcome the decomposition issues associated with oral DSF/Cu formulation. Additionally, the absence of MGMT expression in contralateral brain regions suggests that HPβCD-Cu(DDC)₂ minimizes neurotoxicity59making it a safer alternative for long-term use. Our data confirms that MGMT degradation occurs through the ubiquitin-proteasome pathway. Proteasome-targeted therapies can provide new avenues for treating other MGMT-expressing tumors beyond GBM60. While our study focused on TMZ, the HPβCD-Cu(DDC)₂ system could potentially enhance the efficacy of other alkylating agents like lomustine (CCNU) which is the standard second line agent for GBM and carmustine (BCNU). This creates a tremendous opportunity for further investigation into combination therapies for different cancer types with MGMT overexpression. TMZ and CCNU are the primary treatment for the majority of malignant primary brain tumors and would have implications for astrocytoma and oligodendroglioma tumors61. The encouraging preclinical results warrant further investigation through Phase I/II clinical trials to validate the safety and efficacy of this delivery system in humans. Future research could also explore alternative cyclodextrin formulations or additional modifications to improve BBB penetration and drug release dynamics.

Our in vivo studies using PDOX derived from MGMT-positive patient tumors demonstrated that intravenous administration of HPβCD-Cu(DDC)₂ with TMZ led to significant tumor size regression compared to TMZ alone (Fig. 7). This result is in consistent with previous report which emphasized that multimodal therapy combining drug delivery systems with chemotherapy can overcome resistance and improve outcomes62. Importantly, in our model, the HPβCD vehicle alone had no anti-tumor effect, confirming that the observed efficacy is due to the synergistic effct of HPβCD-Cu(DDC)₂ and TMZ. It has been demonstrated that HPβCD vehicle has no therapeutic effect on HF2303 tumor growth22. Our findings also show that HPβCD-Cu(DDC)₂ prevents MGMT expression in tumors without affecting contralateral brain tissues (Fig. 8). This localized inhibition minimizes off-target toxicity, which has been a challenge for other drug delivery systems. This supports earlier research by Narsinh et al.., who highlighted the importance of selective delivery to minimize systemic side effects in GBM treatments63. These findings strongly suggest that the synergistic interaction between HPβCD-Cu(DDC)₂ and TMZ enhances the antitumor effect, providing a promising strategy to address the clinical challenge of TMZ resistance in GBM patients. Additional mechanistic studies are warranted to explore the underlying pathways and validate these findings in clinical settings.

Despite the promising outcomes, some limitations must be addressed. First, although HPβCD-Cu(DDC)₂ demonstrated tumor size regression in our PDOXs, tumor recurrence remains a significant challenge in GBM. Combining this delivery system with immunotherapeutic modulation approaches or targeted inhibitors may yield more durable responses. Future studies could focus on optimizing the dosing schedule and exploring nanoparticle-mediated delivery methods to further improve target efficiency. Additionally, examining the impact of HPβCD-Cu(DDC)₂ on the tumor microenvironment can provide valuable insights into how this delivery system influences immune cell infiltration and angiogenesis.

Conclusion

In summary, the present study introduces a novel HPβCD-Cu(DDC)₂ delivery system that enhances TMZ efficacy by targeting MGMT-mediated resistance in GBM. This system offers a promising new therapeutic approach for patients with limited treatment options. We have succeeded in delivering HPβCD-Cu(DDC)₂ to GBM cells, our strategy can revolutionize treatment option for GBM and other MGMT-expressing malignancies. Further clinical investigation is warranted to confirm the safety and efficacy of this approach in human trials. The potential to extend this delivery platform to other drugs and cancer types underscores its broad applicability and transformative potential in GBM.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (344.3KB, pdf)
Supplementary Material 2 (261.6KB, pdf)
Supplementary Material 3 (590.7KB, docx)
Supplementary Material 4 (642.2KB, docx)
Supplementary Material 5 (690.7KB, docx)

Author contributions

Conceptualization, M.M.A. and M.A.R.; writing-original draft, M.A.R., M.K.Y. M.M.A; visualization and writing-review M.M.A; synthesis and experimental design, H.S, S.G and M.K.Y; editing J.R.E, J.M.S, A.D and M.M.A; supervision; project administration; and funding acquisition; M.M.A. All authors have read and agreed to the published version of the manuscript.

Funding

The authors acknowledge research support from the National Institutes of Health (NIH) grant RO1CA269607 to MMA.

Data availability

The research data used in this study can be shared upon reasonable request sent to the corresponding authors.

Declarations

Competing interests

The authors declare no competing interests. Henry Ford Health has received travel compensation on behalf of JMS for presentations not related to this work by Hoffmann-La Roche AG and Premier Applied Sciences.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (344.3KB, pdf)
Supplementary Material 2 (261.6KB, pdf)
Supplementary Material 3 (590.7KB, docx)
Supplementary Material 4 (642.2KB, docx)
Supplementary Material 5 (690.7KB, docx)

Data Availability Statement

The research data used in this study can be shared upon reasonable request sent to the corresponding authors.

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