Final Product€¦ · Final Product Introduction Glioblastoma (GBM) is the most common primary malignant brain tumor in adults, accounting for approximately 39% of central nervous

Final Product

============================== MetaData ============================== Journal: Journal of Cancer Research and Discovery(JCRD) Article Type: Basic Research Article Title: Microbiological analysis of endotracheal aspirate and endotracheal tube cultures in mechanically ventilated patients Short Title: Microbiological ana endotracheal Authors: Zheng Chen (1 3); Xiangyu Q. Wei (3); Xuesheng Zheng (1 2); Affiliations: 1: At the same time, Pseudomonas aeruginosa, normal respiratory flora 2: But results of endotracheal tube and endotracheal 3: The main pathogens cultured in the catheter end culture

Abstract: Abstract Objective To analyze the consistency of microbiological culture results between endotracheal aspirate and endotracheal tube end specimens in ICU patients with mechanical ventilation. Method From January 2017 to December 2017 a total of 81 patients undergoing mechanical ventilation in the[a] intensive care unit were continuously enrolled. The results of endotracheal tube and endotracheal aspirate cultures were recorded and analyzed. Spearman correlation analysis was used to analyze the relevance of the results of the twospecimens. The consistency of the results of the two specimens was analyzed by Kappa analysis and principal component analysis. Results The main pathogens cultured in the catheter end culture and tracheal suction specimens were Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, and pneumonia Klebsiella. The results of spreaman analysis showed a positive correlation between the results of the two specimens [b]. At the same time, Pseudomonas aeruginosa, normal respiratory flora, Staphylococcus aureus, Klebsiella pneumoniae, and Acinetobacter baumannii were also positively correlated in both specimens [c]. Kappa analysis also showed the consistency of the microbiological culture results of the endotracheal aspirates and the end ofcatheters was very high [d]. The K values of Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae and Acinetobacter baumannii were [e], respectively. Meanwhile, the principal component analysis also showed higher similarity. Conclusion The microbiological culture results of tracheal aspiration specimens in patients with ICU mechanical ventilation are similar to those of catheter end cultures. In clinical practice, it has a high reference value as an etiological specimen. Keywords: tracheal aspirate; endotracheal tube end; mechanical ventilation; microorganism; clinical value; consistency analysis Corresponding Author: Xue-sheng Zheng Department of Neurosurgery, Xinhua Hospital, Affiliated to Shanghai Jiao Tong University School of Medicine Address: 1665 Kongjiang Road, Yangpu District, Shanghai 200092, China (PRC) Shanghai Shanghai China (PRC) Tel: 86-15001932566 Corresponding Email: [email protected]

Final Product

Cover Letter

Dear Editor in Chief:

We would like to submit the revised manuscript entitled “FM19G11

inhibits O6-methylguanine DNA-methyltransferase expression under

both hypoxic and normoxic conditions”, which we wish to be considered

for publication in Cancer Medicine.

The authors declare no conflicts of interest.

We have reviewed the final version of the manuscript and approve it

for publication. To the best of our knowledge and belief, this manuscript

has not been published in whole or in part nor is it being considered for

publication elsewhere.

Best Regards.

Yours Sincerely,

Chao-guo You, Han-song Sheng, Chao-ran Xie, Nu Zhang, Xue-sheng Zheng

Final Product

FM19G11 is a low-toxicityinhibits O6-methylguanine

DNA-methyltransferase inhibitor expression under

both hypoxic and normoxic conditions

Final Product

Abstract:

Background: FM19G11 is a small molecular agent that inhibits hypoxia-inducible factor-1-alpha

(HIF-1α) and other signaling pathways. In this study, we characterized the modulating effects of

FM19G11 on O6-methylguanine DNA-methyltransferase (MGMT), the main regulator of

temozolomide (TMZ) resistance in glioblastomas.

Methods: This study included two MGMT-positive cell lines (GBM-XD and T98G). MGMT

promoter methylation status, mRNA abundance, and protein levels were determined before and

after FM19G11 treatment, and the roles of various signaling pathways were characterized.

Results: Under hypoxic conditions, MGMT mRNA and protein levels were significantly

downregulated by FM19G11 via the HIF-1α pathway in both GBM-XD and T98G cells. In

normoxic culture, T98G cells were strongly positive for MGMT, and MGMT expression was

substantially down-regulated by FM19G11 via the NF-κB pathway. In addition, TMZ resistance

was reversed by treatment with FM19G11. Meanwhile, FM19G11 has no cytotoxicity at its

effective dose.

Conclusions: FM19G11 could potentially be used to counteract TMZ resistance in MGMT-

positive glioblastomas.

Keywords: FM19G11; O6-methylguanin DNA-methyltransferase; glioblastoma

Abbreviation list:

O6-methylguanin DNA-methyltransferase (MGMT)

Glioblastoma (GBM)

Temozolomide (TMZ)

Overall survival (OS)

Final Product

Introduction

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults,

accounting for approximately 39% of central nervous system neoplasms. Treatment typically

includes maximal safe resection plus post-operative adjuvant ionizing radiation and

chemotherapy with temozolomide (TMZ). In 2005, Stupp et al. reported a 2.5-month

overall survival (OS) benefit with the addition of the alkylating agent TMZ to surgery and

radiation ; thus, this treatment plan is termed Stupp’s regimen.

Although the median survival time of GBM patients receiving the Stupp regimen is only 14.6

months, it is noteworthy that patients with low O6-methylguanin DNA-methyltransferase

(MGMT) expression benefit more from TMZ chemotherapy than patients with high MGMT

expression . According to a recent study, the median OS of a low-MGMT group of GBM patients

receiving the Stupp regimen was 21.8 months, while that of a high-MGMT group was only 13.1

months .

TMZ exhibits cytotoxicity mostly by methylating the O6 position of guanine and then causing

tumor cell apoptosis. MGMT directly removes the methyl group at the O6 position of guanine and

thus reverses the cytotoxic effects of TMZ . Therefore, inhibition of MGMT expression may help

to overcome TMZ resistance in GBM .

If the MGMT promotor is methylated, the gene is silenced . Otherwise, the gene will be

expressed, and the expression efficiency is modulated by many mechanisms, including the

hypoxia-inducible factor-1-alpha (HIF-1α), NF-κB , and WNT/β-catenin pathways .

Recently, Tang et al. reported that the HIF-1α inhibitor 2-methoxyestradiol (2-ME)

downregulated MGMT expression under hypoxic conditions . However, 2-ME is highly cytotoxic,

which limits further investigation in pre-clinical settings, and it only exerts an effect under

hypoxic conditions . FM19G11 is a novel small molecule (molecular weight: 463.40 g/mol) HIF-

1α inhibitor with an effective dose in the nanomolar range, and it is very safe at concentrations

lower than 30 μM . In addition, FM19G11 modulates other signaling pathways, including mTOR

and PI3K/Akt/eNOS , when the HIF-1α pathway is inactivated under normoxic conditions.

Therefore, we hypothesized that FM19G11 suppresses MGMT expression under both hypoxic and

Final Product

normoxic conditions through different mechanisms. The present study was performed to test this

hypothesis.

Final Product

Methods

GBM cell culture: The T98G GBM cell line was provided and authenticated by the Shanghai

Institute of Biochemistry and Cell Biology, Shanghai, China. GBM-XD is a primary cell strain

derived from the surgical specimen of a patient with World Health Organization grade IV GBM

undergoing resection in accordance with a protocol approved by the Ethics Committee of our

hospital, and with prior informed consent from the patient. The culture medium was composed of

DMEM (Life Technologies/GIBCO, Carlsbad, CA, USA) and 10% fetal bovine serum (FBS; Life

Technologies/GIBCO). The cells were cultured at a density of 1 × 105 cells /ml. For normoxic

culture, the cells were incubated at 37°C with 95% air, 5% CO2, and 100% humidity. For hypoxic

culture, the cells were incubated at 37°C with 1% O2, 5% CO2, and 100% humidity.

Cell viability assay: The cytotoxic effects of TMZ (Sigma-Aldrich, St. Louis, MO, USA) and

FM19G11 (Sigma-Aldrich) were measured using the CellTiter 96 AQueous Non-Radioactive Cell

Proliferation Assay (Promega Corp., Madison, WI, USA) following the manufacturer’s protocol.

Briefly, GBM-XD and T98G cells were seeded in 96-well flat-bottom plates at 5,000 cells/well,

cultured in DMEM supplemented with 10% FBS, and then treated with TMZ and/or FM19G11, or

DMSO as a control. A mixture of 100 µl phenazine methosulfate (PMS) and 2 ml of [3-(4,5-

dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)

reagent was freshly prepared. Next, 20 µl of MTS/PMS mix reagent was added to 100 µl of media

per well, and the cells were incubated at 37°C for 2 h. The optical density (OD) was measured at

490 nm with a spectrophotometer. Relative cell viability was expressed as the ratio of the OD of

TMZ and/or FM19G11-treated cells to the OD of control cells.

Western blotting: Cells were rinsed with phosphate-buffered saline (PBS), lysed in

radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 25 mM Tris [pH 7.4], 1% Triton

X-100, 0.5% sodium dodecyl sulfate [SDS], and 5 mM EDTA), and cleared by centrifugation in a

microfuge at 20,000 × g for 15 min. The protein concentration was determined using bovine

serum albumin as the standard. Equal amounts of protein were used for SDS polyacrylamide gel

Final Product

electrophoresis (SDS-PAGE). Gels were electroblotted onto nitrocellulose membranes that were

blocked for 1 h with 5% non-fat dry milk in tris-buffered saline (TBS) containing 0.1% Tween-20.

The membranes were then incubated at 4°C overnight with primary antibodies. After rinsing in

PBS, the membranes were incubated at room temperature for 1 h with peroxidase-conjugated

secondary antibodies, and then developed using the enhanced chemiluminescence (ECL) system

(Amersham Biosciences, Little Chalfont, United Kingdom). The primary antibodies used were

specific for MGMT, GAPDH, IKBα, IKKα, P65, TCF1, LEF1, -catenin, c-Myc, C-Jun, HIF-1α,

EPO, and VEGF (ABCAM, Cambridge, USA).

Real-time reverse transcription (RT)-PCR: Total RNA from GBM-XD and T98G cells was

extracted using TRIzol reagent (Invitrogen Corp., Carlsbad, CA, USA) according to the

manufacturer’s instructions. RT was carried out with 2 μg of RNA as the template in a total

volume of 20 µl with a RevertAid First Strand cDNA Synthesis Kit (Fermentas, Waltham, MA,

USA). The primer sequences for the MGMT gene were: Forward, 5'-

GTTATGAATGTAGGAGCCCTTATG-3'; and Reverse, 5'-TGACAACGGGAATGAAGTAATG-

3'. The amplicon size was 239 bp. Real-time PCR was performed with the SuperScript III OneStep

RT-PCR System (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s

instructions. The results are expressed as relative mRNA levels (mRNA level in FM19G1-treated

cells/mRNA level in untreated control cells).

Immunofluorescence staining: Tumor cells were cultured on coverslips. After the cells attached to

the slips over 24 h, FM19G11 was added to the culture media for 72 h. The cells were then fixed

with 4% formaldehyde (10 min), permeabilized with 0.1% Triton X-100 for 5 min, and blocked

with 1% BSA/10% normal goat serum for 1 h. The cells were then incubated with anti-MGMT

antibodies (Abcam, Cambridge, USA) overnight at 4°C in the dark, followed by incubation at

room temperature for 1 h with secondary antibodies (Abcam). Then, the slips were rinsed in PBST

once and in PBS twice, and mounted with DAPI mounting solution. The slips were immediately

examined under a fluorescence microscope.

Flow cytometry: T98G cells were dissociated with 0.25% trypsin to prepare a cell suspension. The

Final Product

suspension was filtered through a cell strainer to remove cell clusters. The total cell number was

then counted. The single-cell suspension was centrifuged at 600 rpm for 1–2 minutes at 4°C to

remove the supernatant. The cell pellet was then resuspended in 5 ml of DMEM supplemented

with 10% FBS. TMZ (500 μM) and/or FM19G11 (300 nM) was then added to the cells, followed

by 2 h of incubation at 37°C. The cells were then washed with cold PBS and stained with an

Annexin V-FITC/propidium iodide solution (BD Biosciences, CA, USA). The samples were

analyzed on a flow cytometer (Beckman Coulter, Brea, CA, USA) using a 488-nm excitation

wavelength.

Bisulfite sequencing PCR (BSP): Genomic DNA was extracted from GBM-XD cells using a

Genomic DNA Mini Tissue Kit (Invitrogen Corp.). Sodium bisulfite modification of 2 μg DNA

was carried out using a MethylEasy DNA Bisulfite Modification Kit (Human Genetic Signatures,

Sydney, Australia). The MGMT promoter-associated CpG island is 267 bp in length and contains

27 CpG sites. The following primer pairs were used: F, 5'-GGATATGTTGGGATAGTT-3'; and R,

5'-AAACTAAACAACACCTAAA-3'. The PCR mixture contained 1 μl of each primer, 3 μl of

bisulfite-treated DNA, 0.8 μl of Taq DNA polymerase (4 U), and 200 mM dNTPs in a final

volume of 50 μl. PCR was performed with an initial denaturation step at 98°C for 4 min followed

by 40 cycles of denaturation at 94°C for 45 s, annealing at 56°C for 45 s, and extension for 1 min

at 72°C, and a final extension at 72°C for 8 min. The PCR products were cleaned using a

ChargeSwith PCR Clean-Up Kit (Invitrogen Corp.) and cloned using pUC18-T (Sangon Biotech,

Shanghai, China). The bacterial colonies containing the recombinant plasmid were amplified, and

the plasmid was extracted. The plasmid was then sequenced. Ten clones were sequenced for each

sample.

Statistical analysis: An analysis of variance was used to analyze the cell viability assay data. The

chi-square test was used to analyze the flow cytometry data. P<0.05 was accepted as statistically

significant.

Final Product

Results

Immunocytochemistry and Western blotting showed that both GBM-XD and T98G cells were

MGMT-positive in hypoxic culture (Figures 1 and 2). After FM19G11 treatment (300 nM for 3

days), MGMT expression was significantly suppressed in both cell lines (Figures 1 and 2). In

normoxic culture, T98G cells were strongly positive for MGMT, and MGMT expression was

substantially down-regulated by FM19G11. However, MGMT expression in GBM-XD cells was

weak in normoxic culture, and the effect of FM19G11 on MGMT expression was unobservable

(Figures 1 and 2). Similarly, the mRNA levels of MGMT were significantly down-regulated by

FM19G11 treatment in hypoxic GBM-XD, hypoxic T98G, and normoxic T98G cells (Figure 3).

Since promotor methylation is the main regulator of MGMT transcription, we examined the

methylation status of the MGMT promoter and found that GBM-XD was completely

unmethylated, and that T98G was partially unmethylated, with only 1 of the 27 CpG sites

methylated. After FM19G11 treatment (300 nM for 30 days), the promoter methylation status did

not change in either cell line (Figure 4), suggesting that the effect of FM19G11 on MGMT

expression is unrelated to promotor methylation.

We next explored several signaling pathways that may be involved in the modulation of MGMT

transcription under hypoxic conditions. HIF-1α was expressed in GBM-XD and T98G cells. After

FM19G11 treatment, the levels of HIF-1α and its target genes EPO and VEGF were decreased,

and the change was similar to that of MGMT (Figure 5). For the WNT/β-catenin pathway, the β-

catenin levels remained constant in the absence or presence of FM19G11. In addition, the related

transcription factors TCF1 and LEF1 and the target genes C-JUN and C-MYC changed irregularly

after FM19G11 treatment (Figure 6). For the NF-κB pathway, we found that P65, a member of the

NF-κB family, and the regulatory molecules IKBα and IKKα were unaffected by FM19G11

(Figure 7). Taken together, we conclude that under hypoxic conditions FM19G11 inhibits

MGMT expression mainly via the HIF-1α pathway.

Under normoxic conditions, HIF-1α is inactivated, so we explored only the WNT/β-catenin and

NF-κB pathways. Again, the second messenger β-catenin remained unchanged after FM19G11

treatment, and TCF1, LEF1, C-JUN, and C-MYC changed irregularly (Figure 8). For the NF-κB

Final Product

pathway, P65 decreased dramatically after FM19G11 treatment in T98G cells in normoxic culture,

and the change was similar to that of MGMT (Figure 9). However, the regulatory molecules IKBα

and IKKα did not change. These results suggest that, under normoxic conditions, FM19G11

directly inhibits NF-κB and thus down-regulates MGMT expression.

As shown in Figure 10, the cell viability assay demonstrated that FM19G11 (300 nM for 3

days) had no cytotoxicity by itself. However, FM19G11 significantly enhanced the cytotoxicity of

TMZ (100 µM for 3 days) in hypoxic GBM-XD cells (TMZ group 0.754 ± 0.048 vs.

TMZ+FM19G11 group 0.464 ± 0.015, P<0.05), hypoxic T98G cells (TMZ group 0.498 ± 0.016

vs. TMZ+FM19G11 group 0.339 ± 0.009, P<0.05), and normoxic T98G cells (TMZ group 0.488 ±

0.012 vs TMZ+FM19G11 group 0.327 ± 0.010, P<0.05). Flow cytometry showed that the early

apoptosis percentage was 18.9% in the TMZ group and 26.6% in the TMZ+FM19G11 group

(P<0.05), while the late apoptosis percentage was 27.0% in the TMZ group and 36.8% in the

TMZ+FM19G11 group (P<0.05). These results indicate that FM19G11 significantly enhanced the

pro-apoptotic effect of TMZ, although FM19G11 did not induce apoptosis by itself (Figure 11).

Final Product

Discussion

The alkylating agent TMZ is currently the standard chemotherapy for GBM . As a prodrug, it

undergoes spontaneous decomposition in solution at a physiological pH to the reactive

intermediate 5-(3-methyl-1-triazeno)imidazole-4-carboxamide, which methylates the N7 and O6

positions of guanine and the N3 position of adenine . Among these three methylation positions, O6-

methylguanine (O6-meG) is the most important mismatch; it results in a continuous cycle of DNA

base mismatch repair with eventual strand breaks, ultimately leading to cellular apoptosis, which

is how TMZ exerts its chemotherapeutic effects .

However, if MGMT is expressed in tumor cells it will directly remove the methyl group from

O6-meG, causing the cells to become TMZ-resistant . Every methyl group removed from O6-meG

is transferred to a cysteine residue within the active site of MGMT in a stoichiometric reaction,

and this MGMT molecule is then inactivated and finally degraded. This process of destruction of

one MGMT molecule for each methyl group removed from methylguanine is termed suicide

inhibition . Therefore, TMZ cytotoxicity is theoretically determined by the ratio of MGMT to O6-

meG .

Many different agents have been tested for their ability to inhibit MGMT, with little success .

For example, TMZ itself was shown to partially deplete MGMT protein in tumors. In clinical

practice, a dose-dense TMZ regimen is applied to some recurrent MGMT-unmethylated gliomas ,

based on the idea that a high dose of TMZ would probably deplete MGMT in tumor cells via

suicide inhibition . However, these large clinical trials failed to produce an OS benefit of

increasing TMZ dose intensity . Another limitation of this strategy is that high-dose drug exposure

inevitably leads to increased side effects .

Some other chemotherapeutic agents have been tried to bypass the limitations of TMZ in

MGMT unmethylated patients, including 1,3-bis(2-chloroethyl)1-nitrosourea or 3-[(4-amino-2-

methyl-5-pyrimidinyl) methyl]-1-(2-chloroethyl)-1-nitrosourea hydrochloride. However, the

hematological side effects of these drugs are much more frequent and severe than TMZ .

Another potential method was to sensitize glioma cells to TMZ by concomitant use of the

MGMT pseudosubstrate. One potent agent is O6-BG, a pseudosubstrate inactivator of MGMT.

Final Product

This agent has been shown to reverse resistance to TMZ by decreasing MGMT protein levels in

glioma cells and animal models . However, in a clinical trial, the addition of O6-BG had little

therapeutic effect, and caused grade 4 hematological events in 48% of the patients, halting further

attempts to use this concomitant therapy .

Taken together, the above mentioned agents have a common drawback; namely, severe side

effects, which limits further study. Therefore, it is necessary to search for new agents to suppress

MGMT expression, especially ones with low cytotoxicity.

FM19G11 is a small molecule agent with a molecular weight of only 463.40 g/mol . In this

study, we showed that under hypoxic conditions, FM19G11 significantly inhibited MGMT

expression in GBM cells by modulating the HIF-1α pathway. However, under normoxic

conditions, when the HIF-1α pathway was inactivated, FM19G11 inhibited MGMT expression by

modulating the NF-κB pathway. Since there are considerable oxygen concentration gradients in

the lungs and across the capillaries and tumor tissues, the oxygen levels in the tumor should be

lower than that in the air, which is defined as “normoxic condition” in this in vitro study.

Therefore, under the in vivo micro-environments, HIF-1α pathway may play a more important

role than the NF-κB pathway.

Our findings reveal that FM19G11 by itself was not cytotoxic at its effective dose (300 nM for

3 days), implying that this agent is safe for future clinical use. Whenbut when FM19G11 was

given concomitantly with TMZ, it strongly enhanced the pro-apoptotic effect of TMZ. Therefore,

FM1911 could be a candidate for future pre-clinical testing to counteract TMZ resistance in

MGMT-positive glioblastomas. There is a concern that unselective MGMT inhibition is likely to

make the normal tissues, for example, the hematopoietic tissues, more vulnerable to the

cytotoxicity of TMZ, and this might be a major reason for the failure of the above-mentioned

clinical trials. But since the malignant tumor tissue is generally in hypoxic state (Tang et al.

2016), and the MGMT-inhibition effect of FM19G11 majorly relies on the hypoxia-inducible

factor-1-alpha pathway, we think FM19G11 probably preferentially inhibit MGMT activity in

tumor than in the normal tissues. This indirect MGMT-inhibition strategy may be better tolerable

for the body.

Although both TMZ and FM19G11 are from Sigma-Aldrich, the authors declared that there is

no conflict of interest.

Final Product

Final Product

References

Belanich M et al. (1996) Intracellular Localization and intercellular heterogeneity of the human DNA

repair protein O(6)-methylguanine-DNA methyltransferase Cancer chemotherapy and

pharmacology 37:547-555

Dahlrot RH et al. (2017) Prognostic value of O-6-methylguanine-DNA methyltransferase (MGMT)

protein expression in glioblastoma excluding nontumour cells from the analysis

Neuropathology and applied neurobiology doi:10.1111/nan.12415

El Assar M et al. (2015) FM19G11 reverses endothelial dysfunction in rat and human arteries through

stimulation of the PI3K/Akt/eNOS pathway, independently of mTOR/HIF-1alpha activation

British journal of pharmacology 172:1277-1291 doi:10.1111/bph.12993

Fu D, Calvo JA, Samson LD (2012) Balancing repair and tolerance of DNA damage caused by

alkylating agents Nature reviews Cancer 12:104-120 doi:10.1038/nrc3185

Gilbert MR et al. (2013) Dose-dense temozolomide for newly diagnosed glioblastoma: a randomized

phase III clinical trial Journal of clinical oncology : official journal of the American Society of

Clinical Oncology 31:4085-4091 doi:10.1200/JCO.2013.49.6968

Hegi ME et al. (2005) MGMT gene silencing and benefit from temozolomide in glioblastoma The New

England journal of medicine 352:997-1003 doi:10.1056/NEJMoa043331

Hegi ME et al. (2008) Correlation of O6-methylguanine methyltransferase (MGMT) promoter

methylation with clinical outcomes in glioblastoma and clinical strategies to modulate MGMT

activity Journal of clinical oncology : official journal of the American Society of Clinical

Oncology 26:4189-4199 doi:10.1200/JCO.2007.11.5964

Kaloshi G et al. (2010) Nitrosourea-based chemotherapy for low grade gliomas failing initial treatment

with temozolomide Journal of neuro-oncology 100:439-441 doi:10.1007/s11060-010-0197-6

Kreklau EL, Liu N, Li Z, Cornetta K, Erickson LC (2001) Comparison of single- versus double-bolus

treatments of O(6)-benzylguanine for depletion of O(6)-methylguanine DNA

methyltransferase (MGMT) activity in vivo: development of a novel fluorometric

oligonucleotide assay for measurement of MGMT activity The Journal of pharmacology and

experimental therapeutics 297:524-530

Lan F, Yang Y, Han J, Wu Q, Yu H, Yue X (2016) Sulforaphane reverses chemo-resistance to

temozolomide in glioblastoma cells by NF-kappaB-dependent pathway downregulating

MGMT expression International journal of oncology 48:559-568 doi:10.3892/ijo.2015.3271

Moreno-Manzano V et al. (2010) FM19G11, a new hypoxia-inducible factor (HIF) modulator, affects

stem cell differentiation status The Journal of biological chemistry 285:1333-1342

doi:10.1074/jbc.M109.008326

Nagane M (2015) Dose-dense temozolomide: is it still promising? Neurologia medico-chirurgica

Final Product

55:38-49 doi:10.2176/nmc.ra.2014-0277

Penas-Prado M et al. (2015) Randomized phase II adjuvant factorial study of dose-dense temozolomide

alone and in combination with isotretinoin, celecoxib, and/or thalidomide for glioblastoma

Neuro-oncology 17:266-273 doi:10.1093/neuonc/nou155

Quinn JA et al. (2009) Phase II trial of temozolomide plus o6-benzylguanine in adults with recurrent,

temozolomide-resistant malignant glioma Journal of clinical oncology : official journal of the

American Society of Clinical Oncology 27:1262-1267 doi:10.1200/JCO.2008.18.8417

Rodriguez-Jimenez FJ, Moreno-Manzano V, Mateos-Gregorio P, Royo I, Erceg S, Murguia JR,

Sanchez-Puelles JM (2010) FM19G11: A new modulator of HIF that links mTOR activation

with the DNA damage checkpoint pathways Cell cycle 9:2803-2813

doi:10.4161/cc.9.14.12184

Shirai K, Chakravarti A (2011) Towards personalized therapy for patients with glioblastoma Expert

review of anticancer therapy 11:1935-1944 doi:10.1586/era.11.103

Stupp R et al. (2005) Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma The

New England journal of medicine 352:987-996 doi:10.1056/NEJMoa043330

Tang JH et al. (2016) Downregulation of HIF-1a sensitizes U251 glioma cells to the temozolomide

(TMZ) treatment Experimental cell research 343:148-158 doi:10.1016/j.yexcr.2016.04.011

Terasaki M, Tokutomi T, Shigemori M (2009) Salvage therapy with temozolomide for recurrent or

progressive high-grade gliomas refractory to ACNU [1-(4-amino-2-methyl-5-pyrimidynyl)

methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride] Molecular medicine reports 2:417-421

doi:10.3892/mmr_00000115

van Nifterik KA et al. (2010) Absence of the MGMT protein as well as methylation of the MGMT

promoter predict the sensitivity for temozolomide British journal of cancer 103:29-35

doi:10.1038/sj.bjc.6605712

Weller M, Stupp R, Reifenberger G, Brandes AA, van den Bent MJ, Wick W, Hegi ME (2010) MGMT

promoter methylation in malignant gliomas: ready for personalized medicine? Nature reviews

Neurology 6:39-51 doi:10.1038/nrneurol.2009.197

Wickstrom M et al. (2015) Wnt/beta-catenin pathway regulates MGMT gene expression in cancer and

inhibition of Wnt signalling prevents chemoresistance Nature communications 6:8904

doi:10.1038/ncomms9904

Final Product

Figure legends

Figure 1: Western blotting. Both GBM-XD and T98G cells were positive for MGMT in hypoxic

culture. After FM19G11 treatment (300 nM for 3 days), MGMT expression was significantly

suppressed in both cell lines. In normoxic culture, T98G cells were strongly positive for MGMT,

and MGMT expression was substantially down-regulated by FM19G11. However, MGMT

expression in GBM-XD cells was weak, and the effect of FM19G11 on MGMT expression was

unobservable.

Figure 2: Immunocytochemistry. Both GBM-XD and T98G cells were positive for MGMT in

hypoxic culture. MGMT expression was significantly suppressed in both cell lines by FM19G11.

In normoxic culture, T98G cells were strongly positive for MGMT, and MGMT expression was

substantially down-regulated by FM19G11. However, GMT expression in GBM-XD cells was

weak in normoxic culture.

Figure 3: RT-PCR. The mRNA levels of MGMT were significantly down-regulated by FM19G11

treatment in hypoxic GBM-XD, hypoxic T98G, and normoxic T98G cells.

Figure 4: BSP. The GBM-XD promotor was completely unmethylated, and T98G promotor had

only one CpG site methylated. After FM19G11 treatment (300 nM for 30 days), the promoter

methylation status did not change in either cell line.

Figure 5: Under hypoxic conditions, HIF-1α was expressed in both GBM-XD and T98G cells.

After FM19G11 treatment, the levels of HIF-1α and its target genes EPO and VEGF were

decreased, and the change was similar to that of MGMT.

Figure 6: Under hypoxic conditions, the β-catenin levels remained constant in the absence or

presence of FM19G11. In addition, the related transcription factors TCF1 and LEF1 and the target

genes C-JUN and C-MYC changed irregularly after FM19G11 treatment.

Figure 7: Under hypoxic conditions, P65, a member of the NF-κB family, and the regulatory

molecules IKBα and IKKα were unaffected by FM19G11.

Figure 8: Under normoxic conditions, β-catenin remained unchanged after FM19G11 treatment,

while TCF1, LEF1, C-JUN, and C-MYC changed irregularly.

Figure 9: Under normoxic conditions, P65 decreased dramatically after the FM19G11 treatment of

T98G cells, and the change was similar to that of MGMT. However, the regulatory molecules

IKBα and IKKα did not change accordingly.

Final Product

Figure 10: Cell viability assay. FM19G11 (300 nM for 3 days) had no cytotoxicity by itself.

However, FM19G11 significantly enhanced the cytotoxicity of TMZ (100 µM for 3 days) in

hypoxic GBM-XD cells (TMZ group 0.754 ± 0.048 vs. TMZ+FM19G11 group 0.464 ± 0.015,

P<0.05), hypoxic T98G cells (TMZ group 0.498 ± 0.016 vs. TMZ+FM19G11 group 0.339 ±

0.009, P<0.05), and normoxic T98G cells (TMZ group 0.488 ± 0.012 vs TMZ+FM19G11 group

0.327 ± 0.010, P<0.05).

Figure 11: Flow cytometry. The early apoptosis percentage was 18.9% in the TMZ group and

26.6% in the TMZ+FM19G11 group (P<0.05), while the late apoptosis percentage was 27.0% in

the TMZ group and 36.8% in the TMZ+FM19G11 group (P<0.05). In contrast, the early and late

apoptosis percentages for the FM19G11 group did not exceed that of the control.

Final Product

微微微微微微微微微微微微微微微微微微微微微李李李 李李李李李李李李李李

李李李李李李(MVD)李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李MVD 李李李李李李李

李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李“李李

李李李李”李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李

微微 微微微微微微微李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李

李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李(Zhong, 2003 Neurol Res)李李

李李李李李李李李李李李李李“李李李李李李李”李李李李李李李李李李李李李李李李李李李李李李李李 MVD 李李李李李李李李李李

李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李(Zhong & Li, 2011 Neurosurg)李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李

李 1. 李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李

微微 微微微微微微微微微微微微微微李李李李李

李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李

微微微微微微微微微微微微微微微微微微微微微微微微微李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李①李李李

李李李李李李李李李李李李李李李李李李李李李李李李李李李李 1李李②李李李李李李李李李李李李李李李李李李李李李李李李李李李

李李李李李李李李李李李 2李李③李李李李李李李李李李李李李李/李李李李李李李李李李李李李李李李李李李 3李李④李李李李李李

李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李 4李李⑤李李李李李李李李李李李李李李李李李李李李李李李

李李李李李李李李李李李李李李李李李 5李李

Final Product微微微微微微微微微微微微微微微微微

李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李 REZ 李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李

1 2

4 5

1

2

5

4

3

3

Final Product

微微微微微微微微微微微微微微微微微微李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李

李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李

微微微微微微微微微微微微微微微微李李李李李李李李李李李李李李

李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李

李李2mm李李李李李李李李李李李

李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李 15 李李李 BTEP李BAEP 李李李李李李李李李李李李李李

李李李李李李李李李 20%李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李Zhong & Li,

2008 Neurol Res)李

微微微微微微微微微微微微微微微微微

李李李李 800 李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李

微微微微微微微微微微微微微微微微

患患

患患 患患

患患 患患

患患 患患患

患患 患患 患患患 MRI(+)MRI(-)

患患患MVD患患

患患MVD患患 r-患,患患,患患患患

患患患患患

Final Product

李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李(Zhong & Li, 2010 Acta Neurochir;

Zheng & Li, 2010 Acta Neurochir)李李李李李李李李李李李李李李李 REZ 李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李

微微微微微微微微微微微微微微微微 李李李李李李李李李李李李李李李李李李BTEP李AMR李BAEP李李李李李李李李李李李李李 ZLR 李李李李李李李李李

李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李李(Ying & Zhong, 2011 Int J

Surg)李

李1李李李李李李李李李李李李 AMR 李李李

李2李李李李李李李李李李李李 ZLR 李李李

李3李李李李李李李李李李李李李 BTEP 李李李

Final Product

Dear Dr. Zheng:

Manuscript ID CAM4-2018-01-0130 entitled "FM19G11 is a low-toxicity O6-methylguanine DNA-

methyltransferase inhibitor under both hypoxic and normoxic conditions" which you submitted to

Cancer Medicine, has been reviewed. Some revisions to your manuscript have been

recommended. Therefore, I invite you to respond to the comments appended below and revise

your manuscript.

Before submitting your revisions:

1. Prepare a response to the reviewer comments appended below in point-by-point fashion. In

order to expedite the processing of the revised manuscript, please be as specific as possible in

your response and indicate the page numbers in the manuscript where you have addressed each

comment.

2. Prepare a revised manuscript (word document), highlighting the changes you’ve made. Save

this new document on your computer as you will be asked to upload it during the revision

submission process. NOTE: Please be sure to keep in mind reviewer comments and incorporate

your responses within the manuscript. There may well be areas where you disagree; for example,

you may want to write, "A reviewer suggests that... However, I disagree because...". In any case,

please try to address all of the concerns that are raised within the manuscript as carefully and

adequately as possible in order to avoid unnecessary repetition of the revision process.

3. In addition to your revised manuscript with changes highlighted, please also save a “clean”

copy where the changes are not marked.

To submit your revised manuscript:

1. Log in by clicking on the link below

*** PLEASE NOTE: This is a two-step process. After clicking on the link, you will be directed to a

webpage to confirm. ***

https://mc.manuscriptcentral.com/cancermedicine?

URL_MASK=b4b7b616a59a4d28926796a539303eb1

OR

Log into https://mc.manuscriptcentral.com/cancermedicine and click on Author Center. Under

author resources, use the button “Click here to submit a revision”. PLEASE DO NOT SUBMIT YOUR

REVISIONS AS A NEW MANUSCRIPT.

2. Follow the on-screen instructions. First you will be asked to provide your “Response to

Decision Letter”—this is the response to reviewer comments that you prepared earlier.

Final Product

3. Click through the next few screens to verify that all previously provided information is correct.

4. File Upload: Delete any files that you will be replacing (this includes your old manuscript).

Upload your new revised manuscript file with changes highlighted, a “clean” copy of your revised

manuscript file, any replacement figures/tables, or any new files. Once this is complete, the list of

files in the “My Files” section should ONLY contain the final versions of everything. REMEMBER:

figures/tables should be in jpeg, tiff, or eps format.

5. Review and submit: please be sure to double-check everything carefully so that your

manuscript can be processed as quickly as possible.

Deadlines:

Because we are trying to facilitate timely publication of manuscripts submitted to Cancer

Medicine,your revised manuscript should be uploaded as soon as possible. If it is not possible for

you to submit your revision in 2 months, we may have to consider your paper as a new

submission. If you feel that you will be unable to submit your revision within the time allowed

please contact me to discuss the possibility of extending the revision time.

Keep up to date with Cancer Medicine's new content by signing up for the journal's email alerts

at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)2045-

7634/homepage/get_email_alerts.htm

If you feel that your paper could benefit from English language polishing, you may wish to

consider having your paper professionally edited for English language by a service such as Wiley’s

at http://wileyeditingservices.com. Please note that while this service will greatly improve the

readability of your paper, it does not guarantee acceptance of your paper by the journal.

Once again, thank you for submitting your manuscript to Cancer Medicine and I look forward to

receiving your revision.

Sincerely,

Dr. Qingyi Wei

Editor in Chief, Cancer Medicine

[email protected]

Associate Editor Comments to Author:

Associate Editor

Comments to the Author:

(There are no comments.)

Reviewer(s)' Comments to Author:

Final Product

Reviewer: 1

Comments to the Author

This paper describes FM19G11 which is a low-toxicity indirect O6-methylguanine DNA

methyltransferase inhibitor. Most previous attempts at MGMT inhibition involve the use of low

molecular weight pseudo-substrates that directly and irreversibly inactivate this repair protein. It

should be noted that most of these other inhibitors are of low toxicity in the absence of

alkylation stress at the O-6 position of DNA guanine. From a technical point of view a

considerable amount of well executed experimental work has been performed and the actions of

this interesting molecule have been further characterized. The authors demonstrate a potent

down regulation of MGMT expression via the action of this agent on key signaling pathways, and I

believe this data merits publication. However, I strongly feel that the overall strategy present in

this paper needs to be revised as no strong evidence is presented to explain why this MGMT

inhibitory strategy will succeed therapeutically, when in all previous clinical attempts the

successful in vivo inhibition of MGMT has resulted in negative consequences. I believe that the

non-tumor selective inhibition of MGMT rationale expressed in this paper is fundamentally

flawed and all such agents will by their very nature reduce the therapeutic index of agents such

as temozolomide compared to temozolomide alone. To improve the therapeutic utility of

temozolomide (against MGMT expressing low responsive tumors) this molecule would need to

either exert the observed actions in a tumor cell selective manner or require some additional

mechanism to ensure selective or at least preferential tumor delivery so as not to also reduce

normal tissue MGMT activity. The tumor selectivity of guanine O-6 alkylating agents does not

appears to be due to a large toxicity differential resulting from the lesions themselves, but from

differences in the repair of these lesions by MGMT. Tumors which respond to guanine O-6

alkylating agents do so generally because they contain little or no MGMT activity relative to most

normal tissues, and it is this activity differential that accounts for the majority of their tumor

selectivity. That is, the therapeutic target is essentially the MGMT activity deficit/differential and

is only minimally dependent upon differences between normal and tumor cells in their direct

tolerance or sensitivity to the lesions themselves. Several groups initially argued that MGMT

activity simply represented a barrier preventing the persistence of tumor toxic therapeutic

lesions. Thus it was contended that inhibiting host and tumor protective MGMT activity (as you

have) would allow agents such as temozolomide to act in an unimpaired manner and their utility

would be enhanced, and extended to include the treatment of high MGMT activity tumors.

However, clinical trials have repeatedly demonstrated (as mentioned in this paper) that universal

depletion of MGMT activity (by non-tumor selective strategies) also greatly sensitizes normal

tissues as well as tumor tissues, and actually decreases the therapeutic efficacy. This is the effect

that would be expected if the activity were depended upon a MGMT activity differential. That is,

these inhibitors collapse the host/tumor MGMT activity differential upon which efficacy is based.

Therefore, without a selective action on tumor MGMT activity all universal MGMT inhibitory

strategies are expected to meet the same fate. In essence, the universal ablation of MGMT

activity converts guanine O-6 alkylating agents with selective toxicity to a subset of MGMT

deficient tumors to a universal (host and cancer cell) cytotoxin. Therefore, unless you have a

sound rationale or evidence that FM19G11 will exert the observed actions selectively, the

Final Product

potential therapeutic claims should be strongly attenuated.

An additional point that I think needs to be addressed is the actual levels of oxygen the cells

experienced in your hypoxic and normoxic experiments. The levels of oxygen in the air are not

normoxic as far as tissues are concerned. In fact they are hyperoxic as there are considerable

oxygen concentration gradients in the lungs and across capillaries. Tissue oxygen levels are

typically between 15 - 40 µM (equivalent to a gas mixture containing between ~ 1.5 – 4% oxygen

at 1 atmosphere of pressure). Hypoxic tissues contain levels 0 - 4 µM (equivalent to a gas mixture

containing between ~ 0 - 0.4% oxygen at 1 atmosphere of pressure).

Reviewer: 2

Comments to the Author

Dear editors,

thank you for letting me read this paper on FM19G11. The manuscript is well written and

important in its field. In general, I think the paper is suitable for publication.

Nevertheless I have some comments:

- The authors did not test toxicity of FM19G11. "Low toxicity" should therefore not be mentioned

in the title. Missing cytotoxicity at the effective dose does not necessarily warrant clinical safety.

Otherwise the authors have to present more data.

- Furthermore FM19G11 is not a direct inhibitor of MGMT as implied by the title. Its action is

most probably explained by HIF modulation.

both informations in the title are misleading.

The authors should comment if there is any conflict of interest since TMZ and FM19G11 are from

the same company.

Final Product

Final Product

Final Product

Final Product

FM19G11 inhibits O6-methylguanine DNA-

methyltransferase expression under both hypoxic and

normoxic conditions

Chao-guo You 1,2*, Han-song Sheng 2* , Chao-ran Xie 1,2*, Nu Zhang 2, Xue-sheng Zheng 1#

1 Department of Neurosurgery, Xinhua Hospital, Affiliated to Shanghai JiaoTong University

School of Medicine, Shanghai, China.

2 Department of Neurosurgery, The Second Affiliated Hospital of Wenzhou Medical University,

Wenzhou, Zhejiang, China.

* The first three authors (You CG, Sheng HS and Xie CR) contributed equally to this article.

# Correspondence to: Xue-sheng Zheng, Department of Neurosurgery, Xinhua Hospital, Affiliated

to Shanghai Jiao Tong University School of Medicine, 1665 Kongjiang Road, Yangpu District,

Shanghai 200092, China. Email: [email protected]. Tel: 86-21-25078005. Fax:

86-21-52357635.

Short Running Head: FM19G11 modulates MGMT expression

Conflict of interest statement: We have no conflict of interest. Although both TMZ and FM19G11

are from Sigma-Aldrich, we declare that there is no conflict of interest.

Acknowledge: This study is supported by 2 grants from the Science and Technology Commission

of Shanghai Municipality (#134119a9400 and #14DZ1930303, to Xue-sheng Zheng), 1 grant from

Shanghai Jiaotong University (#YG2015MS65, to Xue-sheng Zheng), and 1 grant from the

Science and Technology Commission of Zhejiang Province (#2016C33SA300055 , to Han-song

Sheng). The English in this document has been checked by at least two professional editors, both

native speakers of English. For a certificate, please see:

http://www.textcheck.com/certificate/oc31XF

Final Product

Detailed Reply to Comments

Reviewer(s)' Comments to Author:

Reviewer: 1

Comments to the Author

This paper describes FM19G11 which is a low-toxicity indirect O6-methylguanine DNA

methyltransferase inhibitor. Most previous attempts at MGMT inhibition involve the use of low

molecular weight pseudo-substrates that directly and irreversibly inactivate this repair protein. It

should be noted that most of these other inhibitors are of low toxicity in the absence of

alkylation stress at the O-6 position of DNA guanine. From a technical point of view a

considerable amount of well executed experimental work has been performed and the actions of

this interesting molecule have been further characterized. The authors demonstrate a potent

down regulation of MGMT expression via the action of this agent on key signaling pathways, and I

believe this data merits publication. However, I strongly feel that the overall strategy present in

this paper needs to be revised as no strong evidence is presented to explain why this MGMT

inhibitory strategy will succeed therapeutically, when in all previous clinical attempts the

successful in vivo inhibition of MGMT has resulted in negative consequences.

Reply: Page 11, our statement as to the future clinical use of FM19G11 is strongly attenuated.

“Our findings reveal that FM19G11 by itself was not cytotoxic at its effective dose (300 nM for 3

days), but when FM19G11 was given concomitantly with TMZ, it strongly enhanced the pro-

apoptotic effect of TMZ. Therefore, FM1911 could be a candidate for future testing to counteract

TMZ resistance in MGMT-positive glioblastomas.” Since the in vivo glioblastoma tissue is

generally in hypoxic state (Tang et al. 2016), and the MGMT-inhibition effect of FM19G11 majorly

relies on the hypoxia-inducible factor-1-alpha pathway, we think FM19G11 probably

preferentially inhibit MGMT activity in tumor than in the normal tissues. This indirect MGMT-

inhibition strategy may be better tolerable for the body.

I believe that the non-tumor selective inhibition of MGMT rationale expressed in this paper is

fundamentally flawed and all such agents will by their very nature reduce the therapeutic index

of agents such as temozolomide compared to temozolomide alone. To improve the therapeutic

utility of temozolomide (against MGMT expressing low responsive tumors) this molecule would

need to either exert the observed actions in a tumor cell selective manner or require some

additional mechanism to ensure selective or at least preferential tumor delivery so as not to also

reduce normal tissue MGMT activity. The tumor selectivity of guanine O-6 alkylating agents does

not appears to be due to a large toxicity differential resulting from the lesions themselves, but

from differences in the repair of these lesions by MGMT. Tumors which respond to guanine O-6

alkylating agents do so generally because they contain little or no MGMT activity relative to most

normal tissues, and it is this activity differential that accounts for the majority of their tumor

selectivity. That is, the therapeutic target is essentially the MGMT activity deficit/differential and

is only minimally dependent upon differences between normal and tumor cells in their direct

Final Product

tolerance or sensitivity to the lesions themselves. Several groups initially argued that MGMT

activity simply represented a barrier preventing the persistence of tumor toxic therapeutic

lesions. Thus it was contended that inhibiting host and tumor protective MGMT activity (as you

have) would allow agents such as temozolomide to act in an unimpaired manner and their utility

would be enhanced, and extended to include the treatment of high MGMT activity tumors.

However, clinical trials have repeatedly demonstrated (as mentioned in this paper) that universal

depletion of MGMT activity (by non-tumor selective strategies) also greatly sensitizes normal

tissues as well as tumor tissues, and actually decreases the therapeutic efficacy. This is the effect

that would be expected if the activity were depended upon a MGMT activity differential. That is,

these inhibitors collapse the host/tumor MGMT activity differential upon which efficacy is based.

Therefore, without a selective action on tumor MGMT activity all universal MGMT inhibitory

strategies are expected to meet the same fate. In essence, the universal ablation of MGMT

activity converts guanine O-6 alkylating agents with selective toxicity to a subset of MGMT

deficient tumors to a universal (host and cancer cell) cytotoxin. Therefore, unless you have a

sound rationale or evidence that FM19G11 will exert the observed actions selectively, the

potential therapeutic claims should be strongly attenuated.

Reply: (1) Page 11, our statement as to the future clinical use of FM19G11 is strongly attenuated.

“Our findings reveal that FM19G11 by itself was not cytotoxic at its effective dose (300 nM for 3

days), but when FM19G11 was given concomitantly with TMZ, it strongly enhanced the pro-

apoptotic effect of TMZ. Therefore, FM1911 could be a candidate for future testing to counteract

TMZ resistance in MGMT-positive glioblastomas.” (2) Page 11. We agree with the concern that

unselective MGMT inhibition is likely to make the normal tissues more vulnerable to the

cytotoxicity of TMZ and this might be a major reason for the failure of the previous clinical trials.

But since the malignant tumor tissue is generally in hypoxic state, and the MGMT-inhibition effect

of FM19G11 majorly relies on the hypoxia-inducible factor-1-alpha pathway, we think FM19G11

probably preferentially inhibit MGMT activity in tumor than in the normal tissues.

An additional point that I think needs to be addressed is the actual levels of oxygen the cells

experienced in your hypoxic and normoxic experiments. The levels of oxygen in the air are not

normoxic as far as tissues are concerned. In fact they are hyperoxic as there are considerable

oxygen concentration gradients in the lungs and across capillaries. Tissue oxygen levels are

typically between 15 - 40 µM (equivalent to a gas mixture containing between ~ 1.5 – 4% oxygen

at 1 atmosphere of pressure). Hypoxic tissues contain levels 0 - 4 µM (equivalent to a gas mixture

containing between ~ 0 - 0.4% oxygen at 1 atmosphere of pressure).

Reply: We agree with this comment, and we discussed this difference of oxygen level between in

vivo and in vitro circumstances in Page 11.

“Since there are considerable oxygen concentration gradients in the lungs and across the

capillaries and tumor tissues, the oxygen levels in the tumor should be lower than that in the air,

which is defined as “normoxic condition” in this in vitro study. Therefore, under the in vivo micro-

environments, HIF-1α pathway may play a more important role than the NF-κB pathway. “

Final Product

Reviewer: 2

Comments to the Author

Dear editors,

thank you for letting me read this paper on FM19G11. The manuscript is well written and

important in its field. In general, I think the paper is suitable for publication.

Nevertheless I have some comments:

- The authors did not test toxicity of FM19G11. "Low toxicity" should therefore not be mentioned

in the title. Missing cytotoxicity at the effective dose does not necessarily warrant clinical safety.

Otherwise the authors have to present more data.

Reply: The title is modified as “FM19G11 inhibits O6-methylguanine DNA-methyltransferase

expression under both hypoxic and normoxic conditions”. “Low-toxicity” is removed from the

title.

- Furthermore FM19G11 is not a direct inhibitor of MGMT as implied by the title. Its action is

most probably explained by HIF modulation.

both informations in the title are misleading.

Reply: The title is modified as “FM19G11 inhibits O6-methylguanine DNA-methyltransferase

expression under both hypoxic and normoxic conditions”, so as to avoid this misleading.

The authors should comment if there is any conflict of interest since TMZ and FM19G11 are from

the same company.

Reply: Page 12. Although both TMZ and FM19G11 are from Sigma-Aldrich, the authors declared

that there is no conflict of interest.