Repurposing Ivermectin to augment chemotherapy’s efficacy in osteosarcoma

Abstract

Background

Osteosarcoma is the most frequent malignant bone malignancy and the current treatments are ineffective. Ivermectin, an anti-protozoal drug, has been shown to have anti-cancer activity. This work investigated the potential of repurposing ivermectin to augment chemotherapy’s efficacy in osteosarcoma.

Methods

Proliferation, migration and apoptosis assays were performed in ivermectin-treated osteosarcoma cells. Combination studies were performed. Osteosarcoma xenograft mouse model was established to investigate the in vivo efficacy of ivermectin. Intracellular reactive oxygen species (ROS) and mitochondrial superoxide, membrane potential, ATP, 8-OHdG level, protein carbonylation and lipid peroxidation were determined after ivermectin treatment.

Results

Ivermectin was effective and acted synergistically with doxorubicin in osteosarcoma cells regardless of cellular origin and genetic profiling. This was achieved through suppressing inhibiting growth and migration, and inducing caspase-dependent apoptosis. Ivermectin also significantly inhibited osteosarcoma growth in vivo and its combination with doxorubicin resulted in much greater efficacy than doxorubicin alone. Importantly, the effective dose of ivermectin was clinically feasible and did not cause significant toxicity in mice. Mechanistical analysis showed that ivermectin induced oxidative stress and damage, and mitochondrial dysfunction.

Conclusions

Our findings indicate that ivermectin has utility in treating patients with osteosarcoma, especially those resistant to chemotherapy.

Introduction

Osteosarcoma is the bone tumor that predominantly affects children and young adults. ¹ The clinical management of osteosarcoma includes surgical resection and chemotherapy which has remained essentially unchanged over the past 3 decades. ² Chemotherapy is comprised of cisplatin, doxorubicin, methotrexate and ifosfamide with limited effectiveness particularly in patients with metastatic disease and their poor survival rate is <30%. ³ New treatment options are required to improve the clinical outcomes of osteosarcoma. Given the high attrition rates and substantial costs of new drug discovery and development, drug repurposing that identifies new uses for approved drugs is an alternative therapeutic strategy for cancer treatment due to their known pharmacokinetics profiling. ⁴

Substantial evidence has shown that ivermectin, a broad anti-parasitic drug, is a candidate for repurposing as an anticancer drug. ⁵ Preclinical studies show that ivermectin inhibits breast cancer growth by stimulating cytostatic macroautophagy/autophagy; ⁶ sensitizes ovarian cancer cells to cisplatin by suppressing Akt/mTOR signaling pathway; ⁷ induces cell cycle arrest and apoptosis of HeLa cells via mitochondrial pathway; ⁸ selectively induces apoptosis in chronic myeloid leukemia through inducing mitochondrial dysfunction and oxidative stress; ⁹ and induces autophagy-mediated cell death in glioma cells. ¹⁰ Of note, ivermectin is an inhibitor of cancer stem-like cells which represent a reservoir of disease and potential source of recurrence and relapse ¹¹ and tumor angiogenesis which is required for tumor progression. ¹² However, little is known on the effects of ivermectin in osteosarcoma.

The aim of this study was to investigate the potential of repurposing ivermectin for the treatment of osteosarcoma. We assessed the efficacy of ivermectin alone and its combination with chemotherapeutic drug in multiple pre-clinical osteosarcoma models, and analysed the underlying mechanisms.

Materials and methods

Cell lines, drugs and MTT assay

Six human osteosarcoma cell lines were obtained from the American Type Culture Collection and the Cell Bank of Type Culture Collection of Chinese Academy of Sciences. Cells were authenticated using short tandem repeat DNA profiling (Procell) and examined for mycoplasma contamination using MycoAlert mycoplasma detection kit (Lonza). Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone). Ivermectin and doxorubicin were obtained from Selleckchem and were reconstituted as per manufacture’s recommendation. Cells were treated with ivermectin, doxorubicin alone or ivermectin and doxorubicin combination. After 24 h, treated cells were subjected to MTT analysis (Abcam) and the absorbance at 570 nm was measured on a Spectramax M5 microplate reader.

Proliferation, apoptosis, and migration assays

Proliferation and apoptosis were determined using BrdU proliferation assay kit (Abcam) and Annexin V-FITC apoptosis kit (Biolegend). Migration was assessed using Cell Biolabs CytoSelect cell Migration assay kit. The treatment duration was 72 h for proliferation and apoptosis assays, and 8 h for migration assay. The procedure of these assays is the same as described in our previous study. ¹³

Determination of mitochondrial superoxide and reactive oxygen species

After 24 h drug treatment, cells were incubated with 10 μM CM-H2DCFDA (Life Technologies) for reactive oxygen species (ROS) and 5 μM MitoSox Red (Life Technologies) for Mitochondrial superoxide. Quantification was performed on Spectramax M5 microplate reader (Molecular Devices) at an ex/em of 495/525 nm (for ROS) and an Ex/Em of 510/580 nm (for MitoSox).

Determination of mitochondrial membrane potential

After 24 h drug treatment, cells were incubated with 100 nM TMRE and 10 μM MitoTracker Green (Life Technologies) for 30 min. After incubation, cells were washed twice with complete media containing 100 nM TMRE. Fresh media with 100 nM TMRE was added prior to reading fluorescence (ex/em 495/525 for MitoTracker green and Ex/Em 550/580 for TMRE) on a Spectramax M5 microplate reader. The ratio of the fluorescence signal of TMRE to that of MitoTracker green represents the mitochondrial potential.

Determination of complex activity, ATP, and hydrogen peroxide

After 24 h drug treatment, cells were homogenized using a standard protocol for total cell lysates. Mitochondrial complex activity, hydrogen peroxide and ATP levels were assessed using cell lysates, and were measured using ATPlite luminescence assay kit (Perkin Elmer), Hydrogen peroxide assay kit (Abcam), Complex I enzyme activity microplate assay kit and Complex II enzyme activity microplate assay kit (Abcam). Luminescence was measured with a 1 s integration time (for ATP levels), absorbance was read at 450 nm (for complex I activity), 600 nm (for complex II activity) in kinetic mode, and fluorometric signal was read at Ex/Em = 535/587 nm (for hydrogen peroxide), on a Spectramax M5 microplate reader.

Determination of protein carbonylation and lipid peroxidation

After 24 h drug treatment, protein carbonylation and lipid peroxidation were measured using the Protein Carbonyl ELISA Kit (Enzo LifeSciences) and the Lipid Peroxidation MDA Assay Kit (Abcam) as per manufacture’s protocol. Absorbance was read on a Spectramax M5 Microplate reader at 532 nm (for lipid peroxidation) and 450 nm (for protein carbonylation).

Oxidative DNA damage

After 24 h drug treatment, cells were harvested by trypsinization and DNA was extracted using the DNEasy Mini Kit (Qiagen). 8-OHdG levels were quantified using the OxiSelect Oxidative DNA Damage ELISA Kit (Cell Biolabs) as per the manufacturer’s protocol. Absorbance was read on a Spectramax M5 microplate reader at 450 nm.

Osteosarcoma growth in vivo

The HOS-143B cells were used to establish osteosarcoma subcutaneous model. Animal studies were approved under the guidelines of the Institutional Animal Care and Use Committees of Renmin Hospital of Wuhan University. Animal studies were conducted in accordance with ARRIVE guidelines. SCID mice were housed in a pathogen-free environment under a 12:12 hour light-dark cycle. Cells at 10 million suspended in 100 μl were subcutaneously injected into mice flank and the treatment was initialized when the tumor volume reached 100–130 mm³. The mice were randomly divided into 2 groups (for single drug treatment) or 4 groups (for combination drug treatment) and received intraperitoneal ivermectin at 0.5 mg/kg once per day, intraperitoneal doxorubicin at 2 mg/kg once per week, or the combination of both drugs. Tumor growth was monitored every week using calliper and volume was determined using formula: length × width²/2.

Statistical analyses

A two-tailed unpaired Student’s t-test was used for individual points. Two-way analysis of variance (ANOVA) was used for comparing the results between two groups. Differences were considered significant for p < .05.

Results

Ivermectin increases the sensitivity of osteosarcoma cells to doxorubicin

We first assessed the effect of ivermectin on osteosarcoma cells using MTT assay. A panel of cell lines that cover varying cellular origins and mutations were used to represent in vitro models of osteosarcoma. ¹⁴ Cells were incubated with ivermectin at concentrations from 1 μM to 10 μM for 24 h. As shown in Figure 1(a), ivermectin dose-dependently decreased growth/viability of all tested osteosarcoma cell lines, with maximum inhibitory effect occurring at 10 μM that resulted in 70%–100% inhibition. In addition, U2OS, MG-63, KPD and Saos-2 were more sensitive than HOS-143B and ZK-58. We further found that ivermectin at 1 μM or 2 μM led to ∼50% inhibition. We next assessed the effect of ivermectin and doxorubicin combination in osteosarcoma cells. Doxorubicin at concentrations that led to 30%–40% or 45%–70% inhibition as single drug alone led to 50%–70% or 75%–95% inhibition when it was combined with ivermectin (Figure 1(b)). These results demonstrate that the combination results in better efficacy compared to single drug alone in all tested osteosarcoma cell lines and furthermore that the enhanced combinatory effects are dose-dependent.

Figure 1.

The effect of ivermectin alone and its combination with doxorubicin on growth/viability of multiple osteosarcoma cell lines. (a) Ivermectin at concentration from 1 to 10 μM dose-dependently decreased growth/viability in osteosarcoma cells. (b) The combination of doxorubicin with ivermectin was more effective than a single drug alone in decreasing growth/viability in osteosarcoma cells. Growth/viability was measured by the MTT assay after 24 h drug treatment. B and D indicated 10 μM and 25 μM in all tested cell lines. A and C indicated 2 μM and 5 μM in HOS-143B; 2 μM and 5 μM in ZK-58; 1 μM and 2 μM in Saos-2; 1 μM and 2 μM in KPD; 0.5 μM and 1 μM in MG-63; 1 μM and 2 μM in U2OS. The experiments were performed in duplicates. At least three independent experiments were performed and the results were shown as relative to control. *p < .05, compared to control; #p < .05, compared to doxorubicin.

Ivermectin inhibits proliferation, decreases migration, and induces caspase-dependent apoptosis in osteosarcoma cells

To determine what cellular activity is inhibited by ivermectin, we performed proliferation and apoptosis assays by incubating treated cells with BrdU or Annexin V. We showed that ivermectin at 1 μM, 2 μM and 5 μM inhibited proliferation in HOS-143B cells by 20%–60% and in MG-63 cells by 50%–90% (Figure 2(a)). Ivermectin increased apoptosis in HOS-143 cells by 20%–50% and in MG-63 cells by 35%–70% (Figure 2(b)). A pan-caspase inhibitor Z-VAD-FMK completed abolished the effect of ivermectin in inducing apoptosis in both two cell lines (Figure 2(c)), indicating that ivermectin induces apoptosis through activating caspase pathway. Boyden chamber assay showed that ivermectin at the same concentration decreased migration in HOS-143 cells by 10%–40% and in MG-63 cells by 30%–50% (Figure 2(d)).

Figure 2.

The in vitro anti-osteosarcoma activities of ivermectin. Ivermectin at concentration from 2 μM to 10 μM decreased proliferation, as assessed by BrdU labeling (a); decreased migration as assessed by Boyden chamber assay (b); and induced apoptosis as assessed by flow cytometry of Annexin V (c) in MG-63 and HOS-143B cells. (d) Z-VAD-FMK (10 μM) completely abolished the pro-apoptotic effect of ivermectin (10 μM) in MG-63 and HOS-143B cells. The experiments were performed in duplicates. At least three independent experiments were performed. *p < .05, compared to control.

Ivermectin augments doxorubicin’s efficacy in in vivo osteosarcoma

To investigate whether the anti-osteosarcoma activity of ivermectin observed in cell culturing system is reproducible in in vivo, we established the osteosarcoma model in SCID mice via subcutaneous injection of HOS-143 cells. A week after inoculation, the mice were randomized to receive treatment with vehicle control and ivermectin for 3 consecutive weeks. We administrated 0.5 mg/kg ivermectin which is clinically feasible on the basis of therapeutic dose of ivermectin for treatment of human parasitic infection that ranges between 0.1 and 0.4 mg/kg. ¹⁵ Throughout the whole duration of treatment, we did not observe any toxicity in mice. Of note, ivermectin significantly decreased osteosarcoma growth beginning at 1st week of treatment. Tumor volume was 40% less in the cohort of mice treated with ivermectin compared with control at 3rd week (Figure 3(a) and (b)). Combination studies showed that although ivermectin and doxorubicin alone inhibited tumor growth, tumor continued to grow to comparable size as control at 4th week (in mice receiving ivermectin) and 7th week (in mice receiving doxorubicin). In contrast, the combination of ivermectin and doxorubicin remarkably arrested osteosarcoma tumor growth and tumor at 8 week was only 1/3 of control tumor in size (Figure 3(c)). Collectively, our data clearly demonstrate that (1) ivermectin inhibits in vivo osteosarcoma growth; (2) the combination results in much better efficacy than doxorubicin alone in mice.

Figure 3.

The efficacy of ivermectin and the synergism between ivermectin and doxorubicin in in vivo osteosarcoma model. (a and b) Representative of tumors isolated from mice treated with control and ivermectin. Ivermectin significantly decreased osteosarcoma growth in mice. (c) Tumor volume measured at the indicated timepoints in control, ivermectin, doxorubicin alone and combination of ivermectin and doxorubicin. *p < .05, compared to control; #p < .05, compared to doxorubicin.

Ivermectin induces oxidative damage and mitochondrial dysfunction in osteosarcoma

Our mechanism studies showed that ivermectin at 2 μM–10 μM significantly elevated intracellular ROS levels in a dose-dependent manner in MG-63 and HOS-143 cells (Figure 4(a)). Hydrogen peroxide, a form of ROS, was elevated in osteosarcoma cells after ivermectin treatment (Figure 4(b)). Mitochondria are main resources of cellular ROS and superoxide anions are the most abundant ROS in the mitochondria. ¹⁶ Consistently, we found that ivermectin increased level of mitochondrial superoxide (Figure 4(c)). The levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG), an oxidized DNA by-product; protein carbonyls, a modification of proteins due to oxidative damage; and malondialdehyde (MDA), an end product of lipid peroxidation, were all elevated in osteosarcoma cells after ivermectin treatment (Figure 4(d) to (f)). These results show that ivermectin induces oxidative stress and damage.

Figure 4.

Ivermectin induces oxidative stress and damage in osteosarcoma cells. Ivermectin increased intracellular ROS (a), hydrogen peroxide (b) and mitochondrial superoxide (c) in MG-63 and HOS-143B cells. Results were shown as relative to control (set up as 1). Ivermectin increased 8-OHdG (d), carbonyls (e) and MDA (f) levels in MG-63 and HOS-143B cells. The experiments were performed in duplicates. At least three independent experiments were performed. *p < .05, compared to control.

Mitochondrial superoxide is generated by leakage of electrons from the mitochondrial electron transport chain to oxygen. ¹⁷ We thus measured mitochondrial functions in MG-63 and HOS-143B cells and found that ivermectin significantly decreased mitochondrial membrane potential and complex I activity (Figure 5(a) and (b)). Ivermectin did not affect activity of mitochondrial complex II (Figure 5(c)), which is consistent with the previous work. ¹² As expected, ivermectin decreased ATP level in osteosarcoma cells (Figure 5(d)). To investigate whether mitochondrial dysfunction induced by ivermectin is the result of apoptosis, we examined mitochondrial function in the presence of Z-VAD-FML. We found that Z-VAD-FMK did not affect ivermectin’s ability in decreasing mitochondrial membrane potential and ATP production (Figure 5(e) and (f)).

Figure 5.

Ivermectin inhibits mitochondrial function in osteosarcoma cells. (a) Ivermectin decreased mitochondrial membrane potential in MG-63 and HOS-143B cells. Ivermectin decreased activity of complex I (b) but not complex II (c) in MG-63 and HOS-143B cells. (d) Ivermectin decreased ATP level in osteosarcoma cells. Z-VAD-FMK did not affect the effects of ivermectin mitochondrial membrane potential (e) and ATP level (f). The experiments were performed in duplicates. At least three independent experiments were performed. *p < .05, compared to control.

Discussion

This work provides the first evidence that ivermectin at clinically feasible dose has activity in osteosarcoma, a malignant disease that the survival rate has not been improved since 1980s. ¹⁸ Ivermectin is an approved drug widely used to treat infections of onchocerciasis and gastrointestinal parasites but has been recently identified to be a candidate of anticancer drug. ¹⁹ Ivermectin is one of the promising drugs among many in development under the repurposing approach because it is safe with mild and self-limiting adverse effects and the effective anticancer concentration is clinically feasible.^20,21

Juarez et al.’s work highlights that ivermectin is a wide-spectrum cancer drug as it inhibits growth of more than 50 human cancer cell lines by some degree. ²¹ In line with the previous work, our findings show that ivermectin is active against multiple osteosarcoma cell lines (Figure 1). Consistent with the fact that osteosarcoma is characterized with high level of molecular and cellular heterogeneity, ²² we observed the varying sensitivity of different osteosarcoma cell lines to ivermectin. Based on human therapeutic doses of ivermectin that range between 0.1 and 0.4 mg/kg which is equivalent to 0.5–2 μM, the concentrations of ivermectin used in osteosarcoma cell culturing models are clinically relevant which is important for drug repurposing. Of note, ivermectin at 1 or 2 μM significantly augmented the inhibitory effect of doxorubicin in all tested osteosarcoma cell lines (Figure 1(b)). A phase I pharmacokinetic study suggests that ivermectin up to 2 mg/kg is tolerable. We also demonstrated that ivermectin at 0.5 mg/kg did not cause toxicity in mice whereas significantly inhibited osteosarcoma growth (Figure 3(a) and (b)). The synergism between ivermectin and doxorubicin was further confirmed in subcutaneous mouse osteosarcoma model. The remarkable and long-lasting inhibition of combination was achieved at the dose of doxorubicin that did not cause significant toxicity in mice (Figure 3(c) and data not shown). The ability of ivermectin in reducing systemic cytotoxicity and enhancing efficacy of chemotherapeutics demonstrates great translational potential of ivermectin for the treatment of osteosarcoma. This finding is also supported by the various studies that ivermectin reverses cancer drug resistance and augments the effects of anti-cancer agents.^7,23,24

Better knowledge of mechanisms of drug activities is essential for repurposing. Molecular mechanisms of ivermectin’s action in cancer include inducing autophagy, mitochondrial dysfunction and oxidative damage, inhibiting the WNT-TCF and Akt/mTOR pathway. ⁵ Our findings suggest that ivermectin acts on osteosarcoma cells via inducing oxidative stress and damage as shown by the increased level of ROS, mitochondrial superoxide, hydrogen peroxide, 8-OHdG, protein carbonyls and MDA (Figure 4). Oxidative stress is closely associated with cancer development and has pro- and anti-tumorigenic effects. ²⁵ Excessive oxidative stress disrupts redox balance via exhausting the antioxidant system capacity and leads to irreparable damage and tumor cell death, as shown in our study that ivermectin decreases mitochondrial membrane potential, complex I activity and ATP production (Figure 5). An increasing number of therapeutic strategies are being developed to elevate ROS levels to eliminate cancer cells, and furthermore that ROS-inducing drugs demonstrate cancer selectivity. ²⁶

Conclusion

In summary, we demonstrate that ivermectin displays a potent anti-osteosarcoma activity via inducing oxidative damage and mitochondrial dysfunction. The synergism between ivermectin and doxorubicin make ivermectin an attractive addition to the treatment armamentarium for osteosarcoma.