Avenaciolide Induces Apoptosis in Human Malignant Meningioma Cells through the Production of Reactive Oxygen Species

Abstract

Malignant meningioma has a poor prognosis and there are currently no effective therapies. Avenaciolide is water-insoluble natural organic product produced by Aspergillus avenaceus G. Smith that can inhibit mitochondrial function. In the present study, we investigated the anti-cancer effects of avenaciolide in an isolated human malignant meningioma cell line, HKBMM. In addition, to assess the specificity of avenaciolide, its effects on normal human neonatal dermal fibroblast HDFn cells were also examined. Avenaciolide showed effective anti-cancer activity, and its cytotoxicity in HKBMM cells was greater than that in HDFn cells. The anti-cancer effects of avenaciolide were mediated by reactive oxygen species (ROS)-induced apoptosis, which may have been caused by mitochondrial disfunction. These results suggest that avenaciolide has potential as a therapeutic drug for malignant meningioma.

INTRODUCTION

Malignant meningioma is uncommon, accounting for approximately 1% of all meningiomas, and has a very poor prognosis after diagnosis.¹⁾ Radical removal is difficult to accomplish by surgery, and tumors are mostly resistant to postoperative radiotherapy and general chemotherapy. Target molecules and potential therapeutic drugs have been reported,²⁾ but there are still no effective treatments, including therapeutic drugs.^3,4) Therefore, efforts to identify drugs for malignant meningioma with few side effects are important. Generally, solid tumors increase glucose metabolism to produce energy and reduce mitochondrial respiration in the Warburg effect.⁵⁾ In contrast, grade III malignant meningioma shows relatively high mitochondrial activity, which enhances the expression levels of oxidative phosphorylation complexes compared with low-grade meningiomas.⁶⁾ Thus, targeting the mitochondrial activity of malignant meningioma is a potential method for identifying candidate drugs.

Recently, we screened a total of 294 natural organic compounds, 10 of which demonstrated potent anti-cancer effects against malignant meningioma cells (data not shown). One promising candidate among these 10 compounds was avenaciolide, because of the potency to induce cell death and the several potential abilities as described below. Avenaciolide is a water-insoluble natural organic product produced by Aspergillus avenaceus G. Smith that has been reported to have antifungal, weak anti-bacterial, and anti-mycobacterial activities.^7,8) Avenaciolide also inhibits mitochondrial glutamate transport,⁹⁾ which causes the inhibition of glutamate metabolism in mitochondria.¹⁰⁾ In addition, avenaciolide was also shown to act as a mitochondrial ionophore to induce the efflux of Ca²⁺ and Mg²⁺ ions from mitochondria.^11,12) These complex features of avenaciolide may affect the mitochondrial respiratory chain,¹³⁾ which is associated with reactive oxygen species (ROS) generation.¹⁴⁾ In addition to its effects on mitochondria, avenaciolide was reported as an inhibitor of lipolysis and glucose/fructose utilization.^15–17) Taken together, these versatile effects of avenaciolide can be used for drug therapy of malignant meningioma. In the present study, we investigated the anti-cancer effects of avenaciolide in an isolated human malignant meningioma cell line, HKBMM, and demonstrated ROS-mediated apoptotic anti-cancer effects.

MATERIALS AND METHODS

Isolation of Avenaciolide

Avenaciolide (Fig. 1) was isolated from the culture broths of fungi as described previously.^18,19) The culture broths were extracted with CH₂Cl₂, and the crude extracts were separated by silica gel column chromatography to purify compounds. Avenaciolide was identified by comparing its reported ¹H- and ¹³C-NMR, mass spectroscopy (MS), and specific rotation data.²⁰⁾

Fig. 1. Chemical Structure of Avenaciolide

Cell Culture

Cell culture was performed as reported previously.²¹⁾ Briefly, human malignant meningioma HKBMM cells were maintained in Ham’s F12 medium (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) with 15% fetal bovine serum (FBS; FUJIFILM Wako Pure Chemical Corporation), and human neonatal dermal fibroblast HDFn cells were maintained in Dulbecco’s modified Eagle’s medium (high glucose; FUJIFILM Wako Pure Chemical Corporation) with 10% FBS on plastic 96-well cell culture dishes (Nippon Genetics, Tokyo, Japan) at 37 °C and 5% CO₂. Cells were incubated for at least 24 h to reach 80% confluency for subsequent experiments. All experimental protocols were approved by the Regulations for Biological Research at Tokyo University of Pharmacy and Life Sciences.

Drug Treatment and Cell Viability Assay

Drug treatment and cell viability assay were performed as described previously.²²⁾ For dose-dependent experiments, cells were exposed to 0–240 µM avenaciolide in culture medium for 24 h at 37 °C and 5% CO₂. Cell Counting Kit-8 (CCK-8) cell viability assay (Dojindo, Kumamoto, Japan) was performed in accordance with the manufacturer’s instructions. Absorbance at 450 nm was measured using a microplate reader (Thermo Fisher Scientific, Tokyo, Japan). For inhibition of ROS or apoptosis, HKBMM cells were treated with 5 mM N-acetyl-L-cysteine (NAC; FUJIFILM Wako Pure Chemical Corporation), 20 µM Z-VAD (Funakoshi, Tokyo, Japan), or 100 µM 7-Cl-O-Nec1(Nec1; Abcam, Cambridge, U.K.) 1 h before avenaciolide treatment. Cells were subsequently exposed to 0–240 µM avenaciolide together with NAC, Z-VAD, or Nec1 for 24 h at 37 °C and 5% CO₂, and then CCK-8 assay was performed.

Fluorescent Detection of ROS Production

CellROX Green (Thermo Fisher Scientific) was used to detect ROS production in accordance with the manufacturer’s instructions. Briefly, cells were cultured on glass bottom dishes (AGC, Tokyo, Japan) and pretreated with culture media in the presence or absence of 5 mM NAC for 1 h, and then exposed to 200 µM avenaciolide. One hour later, 5 µM CellROX Green was applied to cells and incubated for 30 min. Cells were washed three times with PBS containing 2 mM CaCl₂ and 2 mM MgCl₂. Fluorescence was observed under an inverted fluorescent microscope (Eclipse Ti-U: Nikon, Tokyo, Japan) equipped with a CMOS camera (Zyla5.5; Andor Technology, Belfast, U.K.). Fluorescence data were collected and processed by NIS-elements software (Nikon). Fluorescent intensity was measured by ImageJ 1.52k software.²³⁾

Statistics

Statistical analysis was conducted in Excel (Microsoft, Redmond, WA, U.S.A.) with the add-in software Statcel (OMS, Tokyo, Japan). Data are expressed as the mean ± standard error of the mean (S.E.M.). Analyses were performed using Student’s t-test and one-way ANOVA post hoc Tukey–Kramer test. p-Values of less than 0.05 were considered significant.

RESULTS

Dose-Dependent Anti-cancer Effects of Avenaciolide

We first examined the dose-dependent anti-cancer effects of avenaciolide against HKBMM malignant meningioma cells (Fig. 2). Avenaciolide did not promote significant cell death at concentrations not more than 120 µM compared with vehicle; however, it showed potent anti-cancer effects at 160 µM or more. In the normal cell HDFn, 200 µM or more showed significant cell death compared with vehicle. In addition, these higher concentrations (160 µM or more) of avenaciolide in HKBMM cells showed significantly lower cell death in normal HDFn cells. These data suggest that avenaciolide has potential as an anti-cancer drug against malignant meningioma.

Fig. 2. Dose-Dependent Anti-cancer Effects of Avenaciolide

Both malignant meningioma HKBMM cells and human neonatal dermal fibroblast HDFn cells were treated with serial concentrations of avenaciolide for 24 h. Avenaciolide showed stronger cell death effects in cancerous HKBMM cells compared with normal HDFn cells. Data are means ± S.E.M. One-way ANOVA post hoc Tukey–Kramer test for all doses compared to vehicle, ^† p < 0.05, ^†† p < 0.01. Student’s t-test for same dose of HKBMM and HDFn, respectively. n = 8, ** p < 0.01, *** p < 0.001.

ROS-Mediated Anti-cancer Effects of Avenaciolide

Next, we investigated the mechanism of avenaciolide-induced anti-cancer effects. Pretreatment with 5 mM NAC significantly attenuated avenaciolide-induced cell death of HKBMM cells at all concentrations (Fig. 3). To observe ROS production by avenaciolide, we applied fluorescent ROS indicator CellROX green to visualize ROS under our experimental conditions (Fig. 4). We observed strong fluorescence of CellROX green by at least 1- to 1.5-h exposure to 200 µM avenaciolide. In addition, NAC pretreatment significantly suppressed avenaciolide-poduced ROS. Taken together, these data suggest that avenaciolide induces ROS production in HKBMM cells and causes cell death.

Fig. 3. ROS Scavenger NAC Prevented Avenaciolide-Induced Cell Death

HKBMM cells were treated with NAC 1 h before avenaciolide treatment and were exposed to both drugs for 24 h. NAC completely prevented the anti-cancer effects of avenaciolide. Data are means ± S.E.M. Student’s t-test, n = 8. ** p < 0.01, *** p < 0.001.

Fig. 4. Detection of ROS Production Induced by Avenaciolide

(A) Representative fluorescent images of 0 and 200 µM avenaciolide, and 200 µM avenaciolide pretreated with 5 mM NAC. (B) Intensity of CellROX green fluorescence. Significant fluorescent intensity was observed in 200 µM avenaciolide-treated (Ave. +) cells, and 5 mM NAC pretreatment prevented the fluorescence (Ave. +, NAC +). Data are means ± S.E.M. One-way ANOVA post hoc Tukey–Kramer test compared to vehivle, ** p < 0.01, and to 200 µM avenaciolide, ^†† p < 0.01, n = 107, 94, and 98 for vehicle, avenaciolide, and avenaciolide with NAC groups, respectively.

Apoptotic Anti-cancer Effects of Avenaciolide

ROS evokes several cell death pathways, including apoptosis, necrosis, and necroptosis, and may affect other types of cell death. We examined whether the inhibition of apoptosis influenced avenaciolide-induced cell death of HKBMM (Fig. 5). Apoptosis inhibitor Z-VAD was applied together with avenaciolide, which showed complete inhibition of avenaciolide-induced cell death. However, necroptosis inhibitor (necroptosis-associated protein RIP1 inhibitor) Nec1 did not affect avenaciolide-induced cell death (Fig. 6; although the statistical significance can be seen at 240 µM of avenaciolide, it is thought to be no physiological meaning on the whole). Taken together, our findings indicated that avenaciolide-induced anti-cancer effects in HKBMM are mediated by apoptosis.

Fig. 5. Apoptotic Effects of Avenaciolide

Apoptosis blocker Z-VAD was applied 1 h before avenaciolide treatment, and cells were exposed to both drugs for 24 h. Z-VAD completely suppressed avenaciolide-induced cell death. Data are means ± S.E.M. Student’s t-test, n = 8. *** p < 0.001.

Fig. 6. Necroptosis Was Not Induced by Avenaciolide Treatment

Necroptosis inhibitor Nec1 was applied 1 h before avenaciolide treatment, and cells were exposed to both drugs for 24 h. Nec1 did not influence avenaciolide-induced cell death. Data are means ± S.E.M. Student’s t-test, n = 8. * p < 0.05

DISCUSSION

In the present study, we investigated the anti-cancer effects of avenaciolide, a natural organic compound produced by Aspergillus avenaceus G. Smith, against malignant meningioma cells, with relatively low cytotoxic effect observed in normal cells. The anti-cancer effects of avenaciolide were mainly caused by ROS-mediated induction of apoptosis. These results suggested that avenaciolide is potentially useful for malignant meningioma treatment.

Cancer cells alter their metabolism; in general, this is associated with increased ROS, which changes the cellular redox balance.²⁴⁾ Because cancer cells also increase their antioxidant capacity to balance the increased ROS, ROS production and inhibitory enzymes are therapeutic targets for anti-cancer chemotherapy,^25–27) e.g., the combination of bortezomib and suberoylanilide hydroxamic acid (SAHA), both drugs have been approved by U.S. Food and Drug Administration (FDA), synergistically induce ROS mediated apoptosis of nasopharyngeal carcinoma,²⁸⁾ therapeutic anti-cancer drug TAS-103 is known to generate hydrogen peroxide to induce apoptosis in the leukemia cell,²⁹⁾ and doxorubicin is also reported to form oxygen free radical in glioblastoma cell.³⁰⁾ In addition to chemotherapy, enzyme-activated prodrug therapy targeted to oxidative enzymes,²⁶⁾ chemodynamic therapy,³¹⁾ sonodynamic therapy,³²⁾ and photodynamic therapy^33,34) have been developed as ROS-mediated cancer treatments. There are two strategies of ROS augmentation in cancer cells by chemotherapy: activation of ROS inducers and inhibition of ROS scavengers. There are three main ROS inducers: hypoxia,³⁵⁾ cellular metabolic defects,³⁶⁾ and endoplasmic reticulum (ER) stress.³⁷⁾ Additionally, several ROS scavengers have also been well reported, including nuclear factor-erythroid 2-related factor 2 (NRF2) and NRF2-regulated antioxidant enzymes,³⁸⁾ glutathione,³⁹⁾ reduced nicotinamide adenine dinucleotide phosphate (NADPH),⁴⁰⁾ tumor suppressor factors,⁴¹⁾ and dietary antioxidant compounds.⁴²⁾ Thus, in our experiment, avenaciolide was thought to activate or inhibit one or more these factors to produce ROS in HKBMM cells. Avenaciolide was reported as an inhibitor of glutamate transport in rat liver mitochondria.⁹⁾ This factor and/or avenaciolide itself also affects mitochondrial glutamate metabolism.¹⁰⁾ In addition to glutamate transport and metabolism, avenaciolide has a potential function as an ionophore of Mg²⁺ and Ca²⁺ in the mitochondrial membrane.^11,12) Furthermore, studies have reported that avenaciolide can affect glucose/fructose utilization and metabolism of amino acid and palmitic acid.^15–17) Taken together, these features of avenaciolide must disturb the mitochondrial respiratory chain¹³⁾ and consequent ROS production.⁴³⁾

In the present study, avenaciolide-mediated ROS production showed anti-cancer effects through apoptosis in HKBMM cells. Excess ROS exposure itself usually induces apoptotic events in cancer cells,^44,45) which mainly consists of three pathways: the death receptor (DR) signaling pathway; the ER pathway; and the mitochondrial pathway.⁴⁵⁾ Although we did not investigate the pathway of avenaciolide-induced apoptosis, avenaciolide influences mitochondrial function, as described above. These features may cause apoptosis via mitochondrial pathways by affecting the mitochondrial respiratory chain.

In addition to its anti-cancer effects, avenaciolide showed lower cell death induction in normal HDFn cells compared with malignant meningioma HKBMM cells. This differing effect of avenaciolide between normal cells and cancer cells may also be explained by the downregulated mitochondrial activity in malignant meningioma.⁶⁾ Because the net mitochondrial activity is higher in normal tissue than in malignant meningioma, it is possible to consider that a concentration of avenaciolide that partially blocks the mitochondrial activity in HDFn cells completely blocks it in HKBMM cells. The other possible mechanism to explain the different effects may the differences in gene expression in each cell type. One study has reported that mutation or loss of the tumor suppressor gene neurofibromatosis 2 (NF2) is a leading cause of approximately 50% of meningiomas.⁴⁶⁾ In addition to NF2, several mutated genes have been reported in non-NF2 meningiomas, including AKT1, KLF4, PIK3CA, SMO, and TRAF7.^47–49) One or several genes and/or their related pathways may directly or indirectly affect the high specificity of avenaciolide to induce apoptosis in malignant meningioma cells.

In conclusion, avenaciolide has potential as a therapeutic drug for malignant meningioma via induction of ROS-mediated apoptosis. Further investigations are required to clarify the detailed mechanism of ROS production and the induction of apoptosis in specific cancer cell types and to increase its efficacy as an anti-cancer drug.

Acknowledgments

This work was supported by a Grant-in-Aid from the Japan Society for the Promotion of Science (KAKENHI 21K05299), and the Center for Human and Animal Symbiosis Science, Azabu University. We thank H. Nikki March, PhD, for editing a draft of this manuscript.

Conflict of Interest

The authors declare no conflict of interest.

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