PAMs inhibits monoamine oxidase a activity and reduces glioma tumor growth, a potential adjuvant treatment for glioma

Background Monoamine oxidase (MAO) A catalyzes oxidative deamination of monoamine neurotransmitters and dietary amines and regulates brain development and functions. Recently, we showed that MAO A mediates the progression and migration of glioma and MAO A inhibitors reduce glioma cell growth. Glioblastoma (GBM) is a common and most malignant brain tumor which is difficult to treat. Temozolomide (TMZ) is the current standard chemotherapy for glioma, but tumors usually become resistant and recur. So far, no effective therapy for TMZ-resistant glioma is available. Natural plant antimicrobial solution (PAMs) is a Chinese herbal medicine which has been used for decades without toxicity and has multiple medical functions including anti- inflammatory effects. Here, we report the effects of PAMs on glioblastoma growth. Methods The growth of TMZ -sensitive (U251S),-resistant (U251R) human glioma cells, and mouse glioma cell line GL-26 were assessed by MTS colorimetric assay, colony formation, and cell migration assays. Male C57BL/6 mice were implanted subcutaneously or intracranial with luciferase-positive mouse glioma GL-26 cells and treated with vehicle; MAO A inhibitor clorgyline (10 mg/kg); TMZ (1 mg/kg); PAMs (48 mg/kg) alone or in combination with TMZ (1 mg/kg) for 14 days. At the end of the treatment, mice were sacrificed, MAO A catalytic activity in tumors was measured, and tumor sizes were determined by imaging and weight. Results These results show that PAMs inhibits MAO A catalytic activity in all three glioma cell lines studied U251S, U251R, and GL-26. PAMs reduced glioma growth and has greater effects in combination with low dose of TMZ than PAMS or TMZ alone in all three cell lines as shown by MTS, colony formation, and cell migration assays. Using the subcutaneous or intracranial GL-26 glioma mouse model, PAMs reduced the tumor growth and MAO A activity, similar to the MAO A inhibitor clorgyline. Combining PAMs with non-toxic dose TMZ increased survival to a greater extent than those of PAMs or TMZ alone. Conclusions This is the first study which suggests that PAMs alone or co-administration with low doses of TMZ may be a potential adjuvant to reduce the toxicity of TMZ and to abrogate drug resistance for the effective treatment of glioma.


Background
It has been shown that monoamine oxidase A (MAO A) is overexpressed in prostate cancer [1], glioma [2] and Hodgkin lymphoma [3], inhibiting MAO A reduces tumorigenesis and metastasis. MAO A is located in the outer membrane of mitochondria, catalyzes oxidative deamination of neurotransmitters or dietary amines and produces H2O2 [4,5]. MAO A inhibitors have been used for decades for the treatment of neuropsychiatric disorders [6].
Glioblastoma (GBM) is the most aggressive form of primary brain tumors with a median survival of 14 months from the time of diagnosis [7,8]. The standard treatments of newly diagnosed glioblastoma (GBM) are surgery, radiation therapy administered concurrently with oral temozolomide (TMZ), and six cycles of adjuvant TMZ therapy [9]. Unfortunately, less than 10% of the 5-year survival rate accounts for high-grade glioma patients treated with the standard radiotherapy and adjuvant chemotherapy [10]. Almost half of all gliomas are neither TMZ responsive nor susceptible to radiotherapy. Further, increasing the dose of TMZ, a DNA alkylating agent, will increase the toxicity to the bone marrow [11]. Thus, better treatment for GBM is urgently needed. Our previous study showed that MAO A inhibitor clorgyline combined with non-toxic dose TMZ reduced the tumor growth and prolong the survival in animal model [2].
Plant Antimicrobial Solution (PAMs) has anti-inflammation effect, was used for wound infection and festering, cell necrosis, dry gangrene and blood circulation obstacles [12] and was approved to be used as a hospital preparation by China Yunnan Food and Drug Administration.
PAMs is a mixture of Chinese herbal medicine consists of plants, including Carthamus tinctorius, Cymbopogon distans, Lithospermum erythrorhizon, and Solanum indicum, and Blumea balsamifera. we have identified two active compounds from PAMs including Hydroxysafflor yellow A (HSYA) in Carthamus tinctorius and Allantoin in Cymbopogon distans. HSYA exhibited anti-cancer and anti-inflammation function [12] while Allantoin has wound healing function [13]. Recently, we found Shikonia from Lithospermum erythrorhizon inhibited MAO A catalytic activity (unpublished data). Using network pharmacology from three database (TCMSP, Batman and YaTCM), we identified 158 compounds from the herb plants present in PAMs which may be the active components. This information will help us purify and identify additional active ingredients in PAMs by HPLC, GC, and Mass Spectroscopy.
Previous studies showed that PAMs inhibits the TNFα/IFN-γ-induced inflammatory cytokines production in HaCaT cells and ameliorates imiquimod-induced psoriasis-like skin inflammation in vivo through inhibiting the translocation of p65 in NF-κB signaling pathways [12]. Our previous studies showed that treatment with MAO A inhibitor increased TNF-α positive cell population in tumors from glioma animal model [2]. Recently, it has been reported that treatment with MAO A inhibitor reduced the expression of the oncogene NF-κB in prostate cancer [14]. Taken together, this data suggests that MAO A inhibitors regulate the inflammatory response to suppress tumor progression. These findings led us to study if PAMs may have similar properties as a MAO A inhibitor.

Cell culture
Human glioma cell lines U251S (TMZ sensitive) U251R (TMZ resistant), and mouse glioma cell line GL-26 with luciferase were provided by Dr. Florence Hofman at the University of Souther California; Glioma cell lines [15] were cultured in 10% fetal bovine serum in Dulbecco's Modified Eagle's Media (Life Technologies, Carlsbad, CA, USA) supplemented with 100 U/ml penicillin and 0.1 mg/ml streptomycin in a humidified incubator at 37°C and 5% CO2.
MAO a catalytic activity assay MAO A catalytic activity was determined by radioassay as described previously [1,3]. Human prostate cancer LNCaP cells and mouse glioma GL26 cells were used. Clorgyline (Sigma-Aldrich) was pre-incubated with cells at 37°C for 20 min, then the substrate 1 mM 14 C-5-hydroxytryptamine (5-HT) was added, incubated for 20 min at 37°C. At the end of the incubation, the reaction product was extracted, and the radioactivity was determined by the scintillation counter (LS 6500, Beckman coulter, Inc., CA, USA).

MTS assay
Glioma cells U251S, U251R, or GL-26 (5 × 10 3 cells/well) were seeded in 96-well plates and treated with PAMs at various concentrations (0-150 μg/ml) for 48 h. Cell proliferation was determined by an MTS assay kit (Promega, WI, USA). Briefly, MTS reagent (20 μl/well) was added into each well and incubated for 4 h in the presence of 5% CO2 at 37°C. The plate was gently agitated prior to colorimetric analysis, the reaction product of MTS was measured at 490 nm and quantified using a microplate reader Synergy HTX (Bio-Tek, Winooski, VT, USA). Data was plotted using GraphPad Prism (GraphPad Software, San Diego, CA, USA). For the combined treatment with TMZ (Sigma-Aldrich), 5 × 10 3 glioma cells were seeded per well in triplicate and pre-treated with TMZ (15 μM) for 48 h followed by incubation of PAMs and TMZ for 48 h. All experiments were repeated at least three times.

Colony forming assay
Glioma cells were seeded in 96-well plates, various concentrations of PAMs with or without TMZ (15 μM) were incubated for 48 h. After 48 h, cells were re-seeded 500 cells/per well with fresh medium in duplicate. Cells were incubated for an additional 8 to 10 days; colonies were visualized by staining with 1% methylene blue in methanol for 4 h and quantified. Colonies were enumerated from pictures of plates using software tools (open CFU).

Animal models
Male 4-to 6-weeks-old C57BL/6 mice were purchased from Harlan (Indianapolis, IN), house in the animal research facility at University of Southern California (USC), fed with a normal diet. All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC No. 20212) of USC. Mice were sacrificed by carbon dioxide (CO2). For subcutaneous model, Xenograft tumors were established via subcutaneous inoculation of 5 × 10 3 luciferase-positive mouse glioma GL-26 cells into the flank of the mice. Six days after the inoculation, mice were subcutaneously injected with clorgyline (10 mg/kg), PAMs (48 mg/kg) and vehicle respectively for 14 days. Subcutaneous injections were administered in the space between the skin and the underlying muscles over the shoulders.
For intracranial model: mice were treated with (1) PAMs (2) TMZ (1 mg/kg) alone, (3) TMZ and PAMs combination for 14 days. PAMs was injected daily intranasally (IN). TMZ was dissolved in water and administered by gavage for 10 days. Combination of TMZ and PAMs were given with the same schedule as each agent alone.

Statistical analysis
All data was presented as the mean ± standard error (SE) values and analyzed using GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA). T-test was performed for comparison with multiple groups. In brief, we analyzed the data using Prism (two samples and paired) by t-test analysis. A p-value of p < 0.05 was considered statistically significant.

PAMs inhibits MAO a catalytic activity in glioma cells
The effect of PAMs on the inhibition of MAO A catalytic activity was determined in mouse glioma GL-26 cells [2] and human prostate LNCaP cells which have been reported to express high MAO A activity [1]. Our results showed that IC50 of PAMs is 80.0 μg/ml in GL-26 cells and 112.1 μg/ml in LNCaP cells (Fig. 1a). These results indicate that PAMs inhibits MAO A catalytic activity in both glioma and prostate cancer cell lines with almost similar potency.

The effect of PAMs on the growth of tumors derived from GL-26 glioma cells intracranially implanted in C57BL/6 mice
Mice were intracranially implanted with GL-26 glioma cells having luciferase activity. Six days later, mice were treated with PAMs (48 mg/kg) daily intranasally for 14 days. PAMs reduced the brain tumor size (Fig. 5a) and tumor weight compared to vehicle (* p < 0.05, Fig. 5b). MAO A activity was significantly reduced in PAMs treated brain tumor (25%) compared to the vehicle treated (*, p < 0.05, Fig. 5c). PAMs significantly increased survival (19 days) compared to vehicle (16 days) (Fig. 5d). The current standard chemotherapeutic for glioma is TMZ, whose efficacy is Fig. 2 PAMs reduced cancer cell growth in human glioma TMZsensitive (U251S), TMZ-resistant (U251R) and mouse GL-26 cells as determined by colony formation assay. a PAMs reduced cell growth as determined by colony formation in glioma cell lines (U251S, U251R and GL-26). 500 cells were seeded in each well, treated with PAMs for 48 h, incubated for additional 8-10 days, cells were fed every two days. b Cells were treated by PAMs (50 μg/ml), TMZ (15 μM), or PAMs (50 μg/ml) + TMZ (15 μM). Combination of PAMs with low dose TMZ significantly inhibited cell growth as shown by colony formation assay. Data represent the mean ± SEM. The p-value was calculated by t-test. *p < 0.05, **p < 0.01, compared to untreated control (0 μg/ml). The untreated control group was taken as 100%. After combined treatment of PAMs and TMZ, percentage of colonies were 30, 60, and 60 (colony size> 100 mm) in U251S, U251R, and GL-26 cells respectively. Experiments were performed in triplicate and repeated three times with similar results (n = 3)

Li et al. BMC Complementary Medicine and Therapies
(2020) 20:252 Page 6 of 12 accompanied by toxicity and the onset of resistance. This result suggests that PAMs may be used to treat glioma without toxicity. Prolonged treatment with TMZ did not significantly affect tumor growth based on the tumor size (Fig. 6a) and tumor weight (Fig. 6b). Taken together, PAMs or PAMs in combination with TMZ significantly reduced tumor size and weight (* p < 0.05, Fig. 6a and b). Although, combined treatment had the same effect as PAMs alone; but the survival was increased significantly than TMZ or PAMs alone (Fig. 6c). MAO A activity was also inhibited by PAMs alone or in combination with TMZ (Fig. 6d). PAMs decreased tumor growth and prolonged the survival of mice bearing invasive glioma brain tumors. This finding suggests that PAMs reduces tumor growth and increases survival in vivo. In these experiments, we used non-toxic low dose TMZ (1 mg/kg), which by itself has no significant effect on tumor size (Fig. 6a) or weight (Fig. 6b).

Discussion
Increased MAO A has been reported in several types of solid tumors including prostate cancer [1,16], glioma [2], renal carcinoma [14] and lymphoma [3]. MAO A inhibitors have been shown to reduce glioma in both cell and animal models [2]. MAO A inhibitors and its conjugate reduced the prostate cancer progression and metastasis [1], and suppressed cell growth in glioma [2] and Hodgkin lymphoma [3]. These work underscore the significance of targeting MAO A for cancer therapy. The natural plant antimicrobial solution PAMs, which contains multi-bioactive components extracted from Chinese natural and folk medicinal plants has been used clinically in China for hundreds of years. Its effective formula has been shown to prevent inflammation via NF-κB signaling pathways. Recent work showed that PAMs inhibits TNF-α/IFN-γ-induced inflammatory cytokines production and inhibits the translocation of p65 in NF-κB signaling pathways [12,17,18].
The tumor microenvironment of glioma is largely comprised of inflammatory molecules that affect neoplastic process, proliferation, survival and migration of tumors through inflammation and oxidative stress pathways [19]. It has been suggested that NF-κB activation in cancers may be the result of either exposure to proinflammatory stimuli in the tumor microenvironment or mutational activation of upstream components in IκK-NF-κB signaling pathways [17]. It provided evidence that NF-κB is a key transcription factor for the proliferation and survival of glioma cells [20,21].
This study shows the potential use of PAMs for the treatment of glioblastoma via MAO inhibition.
Multiple functions of PAMS have been reported. We have shown previously the antimicrobial effects of PAMs, on Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia. coli, Canidia Albicans, and Aspergillus niger [22]. PAMs remarkably inhibits the growth of Staphylococcus aureus and enhance the wound-healing by increasing the permeability of bacterial cell membranes, leakage of contents, and eventually the death of Staphylococcus aureus. PAMs reduced liver cancer growth by regulating apoptosis in HepG2 cells [23]. Also, we showed the anticancer effect of PAMs in leukemia cells was mediated by anti-proliferation [24]. Recently, we eported that PAMs could also inhibit the tumor growth of cancers by downregulating the expressions of inflammation and vascular growth associated with TNF-α and VEGF [12].
Our studies showed that treatment with MAO A inhibitor increased TNF-α positive population in tumors from animal models [2]. Recently, it has been reported that treatment with MAO A inhibitor reduced the expression of the oncogene NF-κB in prostate cancer [25][26][27][28]. These findings suggest that MAO A inhibitors regulate the inflammatory response to suppress tumor progression [2], and led us to investigate if PAMs may affect MAO A activity.
Here, we demonstrate for the first time that PAMs inhibits MAO A catalytic activity. Further, PAMs reduced the growth of human glioma TMZ-sensitive U251S, TMZ-resistant U251R and mouse glioma GL-26 cells based on MTS, colony formation, and cell migration assays.
Our data shows the IC50 values of PAMs for the growth of U251S, U251R, and GL-26 cells are 115.1, 115.0, 102.4 μg/ml respectively, were 2-fold better than that of clorgyline in all three cell lines. This suggests that herbal medicine mixture PAMs may have more active compounds. The effects of PAMs on normal glia cells are currently under investigation .
TMZ is the current standard therapy for Glioma patients. At least 50% of TMZ treated patients resistant to TMZ. The animal experiments were performed using subcutaneously implanted GL-26 glioma cells in mice. Treatment with PAMs significantly inhibited tumor size, similar to the MAO A specific inhibitor clorgyline. PAMs was also used to treat C57BL/6 mice after they were intracranially implanted with GL-26 glioma cells. The result showed that PAMs significantly inhibits GL-26 tumor growth in their size and weight (~57%). Compared with the vehicle group, PAMs reduced tumor MAO A activity by 25% and improved survival tsignificantly.
We also demonstrated that combining PAMs with low dose of current standard treatment of TMZ was more effective. For the in vivo study, low dose TMZ (1 mg/kg) was used which was lower than the dose administered to patients, non-toxic [2], and no effect on the treatment of glioma. Combining PAMs with low dose TMZ decreased tumor MAO A activity to a greater extent than PAMs or TMZ alone. More importantly, the combination treatment increased survival significantly than either agent alone. TMZ alone had no effect on survival. Thus, PAMs enhances the efficacy of non-toxic low doses of TMZ. This is consistent with our previous finding that MAO A inhibitor clorgyline or the near-infrared-dye conjugated clorgyline (NMI) in combination with TMZ inhibits tumor growth more effectively than each agent alone [2]. Taken together, our findings suggest that PAMs inhibits MAO A activity, has greater effect in combination with non-toxic dose TMZ against glioma, and improves survival in vivo. This finding is consistent with our previous studies showing that knock-down (KD) or pharmacological inhibition of MAO A in prostate cancer and glioma reduces cancer progression [1,2]. Hence, the results show PAMs inhibits MAO A activity and may be used for glioma treatment.

Conclusions
This is the first study showing that the natural plant antimicrobial solution PAMs has MAO A inhibitory effect and suppresses glioma progression. PAMs has been used to treat skin inflammatory diseases and has effect on pain-releasing and wound healing. Here, we show the potential use of PAMs in combination ttherapy with non-toxic dose of TMZ for drug-sensitive and drugresistant gliomas.

Funding
This work was supported by the Tsai family Fund and Boyd-Elsie Welin Professorship to Dr. Jean Shih who is the principal investigator in these grant. Taipei Medical University, Taiwan (03G0000004A) provided Shih-Yin Chen's stipend.
Availability of data and materials All data generated or analyzed during this study are included in this published article.

Ethics approval and consent to participate
The experimental protocol was established, according to the ethical guidelines and was approved by the Institutional Animal Care and Use Committee (IACUC) of University of Southern California (IACUC protocol number 20212). The detail of mice euthanasia methods is following the IACCU guideline, anesthesia with CO2 or isoflurane (1-3% inhalant to effect) will be used for euthanasia of mice. Isoflurane is the standard inhalant anesthetic, delivered to effect in concentrations of 1-3% in oxygen (up to 5% for initial induction), using a precision vaporizer. After surgery, the anesthetic monitoring of mice includes testing of rear foot reflexes before any incision is made, and continual observation of respiratory pattern, mucous membrane color, and responsiveness to manipulations and rear foot reflexes throughout the procedure. This research is not an application for clinical research, an institutional review board (IRB) is not applicable.