Gene

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Gene 776 (2021) 145445

Research paper
ImageMolecular biological investigation of temozolomide and KC7F2 combination in U87MG glioma cell line
Zaka Abbaszade , Bakiye Goker Bagca , Cigir Biray Avci
Kazımdirik, Ege Ünv. Hst. No:9, 35100 Bornova/Izmir, Turkey

A R T I C L E I N F O

Keywords:
Glycolysis
Warburg hypothesis Temozolomide KC7F2
U87 MG
Glioblastoma

A B S T R A C T

Glioblastom Multiforme (GBM) is the most invasive and malignant member of the IV grade of the subclass As- trocytoma according to the last assessment of the 2016 WHO report. Due to the resistance to treatment and weak response, as well as the topographical structure of the blood brain barrier, the treatment is also difficult due to the severe clinical manifestation, and new treatment methods and new therapeutic agents are needed. Temo- zolomide (TMZ) is widely used in the treatment of glioblastoma and is considered as the primary treatment modality. TMZ, a member of the class of cognitive agents, is currently considered the most effective drug because it can easily pass through the blood brain barrier. Glucose metabolism is a complex energy producing machine that, a glucose molecule produces 38 molecules of ATP after full glycolytic catabolism. According to Otto Warburg’s numerous studies cancer cells perform the first glycolytic step without entering the mitochondrial step. These cells produce lactic acid and make the micro-media more acidic even in aerobic conditions. This phenomenon is attributed to the Warburg hypothesis and either as aerobic glycolysis. Although glycolysis en- zymes are the primary actors of this phenotypic expression, some genetic and epigenetic factors are no exception. We experimentally used KC7F2 active ingredient to target cancer metabolism. In our study, we evaluated cancer metabolism in combination with the effect of TMZ chemotherapeutic agent, examining the effect of two different agents separately and in combination to observe the effects of cancer cell proliferation, survival, apoptosis and expression of metabolism genes on expression. We observed that the combined effect of reduced the effective dose of the TMZ alkylating agent and that the effect was increased and the effect of the combined teraphy is assessed from a metabolic point of view and that it suppresses aerobic glycolysis.

1. Introduction

Gliomas constitute 80% of malignant brain tumors, and they are a very common subtype of primary brain tumors with aggressive, highly invasive and neurological destructive properties. Glioblastoma multi- forme (GBM; WHO Astrocitoma Grade IV) is malign primary brain tumor most common in adults. According to their clinical features GBMs
are classified as primary and secondary. Primary or de novo GBMs con- stitue majority of cases (>% 90) and mostly seen in adults (Ohgaki and Kleihues, 2009).

Although the etiology is not known exactly, it is thought to be a spontaneous type of tumor. However, in family history of GBM patients it was found that glioma was seen in close relatives (Houben et al., 2005). Familial form of this tumor has been described for only %1 cases (Schwartzbaum et al., 2006). Additionally, different factors such as, brain damage (Zhen et al., 2010), Human cytomegalovirus (HCMV), ionizing radiation, electromagnetic waves, heavy metals, pestisides,
polycyclic aromatic compounds can also provide a basis for the onset of glioblastoma (Spinelli et al., 2010). According to epidemiologic data every year GBM cases in North America and Europe are observed 2–3 of every 100.000 people (Verdecchia et al., 2002) and the incidence rate in men and women is 1.26: 1 (Mahvash et al., 2011).

2. Conventional teraphy for glioblastoma

Standard GBM treatment is initiated by surgical resection. However, the curative potential of cytoreductive surgery is greatly compromised by the spreading, infiltrative growth pattern of GBMs. The extent of resection is affected by tumor size, location and patient characteristics (ex. age and patient performance status) (Stummer et al., 2006; West- phal et al., 2003; Stupp et al., 2005). Glioblastoma multiforme is char- acterized with high proliferative activity (Schro¨der et al., 1991). When tumor infiltrates into environmental tissues full resection becomes impossible and in this situation radiotherapy is not always efficient

E-mail address: [email protected] (Z. Abbaszade).

https://doi.org/10.1016/j.gene.2021.145445

Received 15 May 2020; Received in revised form 25 December 2020; Accepted 13 January 2021
Available online 21 January 2021
0378-1119/© 2021 Elsevier B.V. All rights reserved.

(Karcher et al., 2006). Blood-brain barrier makes the treatment harder
Under normoxic conditins in non-cancerous cells, HIF-1α isand tumor cells in hypoxic regions are resistant to radiotherapy (Chang et al., 2007). Thats why surgical resection, followed by chemotherapy and radiotherapy, is mainstay for GBM treatment to the extent possible (Simpson et al., 1993).
DNA alkylating agents are the oldest class of anticancer drugs. They are being used actively and maintain their importance for various cancer types including brain tumors (Augustine et al., 2009; Fisher et al., 2007). Alkylating agents damage DNA by creating different big and small ad- ditions with nucleic acid bases. The most promising therapeutic active substance for brain tumors is Temozolomide (TMZ) (Stupp et al., 2005; Hegi et al., 2008). TMZ is an oral alkylating agent that prevents cancer cell development, slows down their growth and disperses inside the body (Neidle and Thurston, 2005).
TMZ as a prodrug is an imidasole derivative. It is a second generation alkylating chemoterapeutic agent developed 1980s. Since TMZ is lipo- philic, it effectively passes blood- brain barrier (Denny et al., 1994; Tentori and Graziani, 2009).
TMZ leads cell death through alkylation of guanine in O6 position and deterioration of DNA replication (Drabløs et al., 2004). O6-meth- ylguanine-DNA methyltransferase (MGMT), DNA repair protein, plays arole in resistance of tumor cells to alkylating agents (Wedge et al., 1996). MGMT is expressed in gliomas and its contribution to TMZ resistance is known (Wedge and Newlands, 1996; Kyrtopoulos et al., 1997). Current teraphy standart for newly diagnosed GBM patients is surgery, radio- teraphy and adjuvan TMZ teraphy and their survival time is up to 14.6 months, but median survival time is 12.6 months for patients recieving only radioteraphy.

TMZ cytotoxicity is carried out by O6-MeG which is mutagenic, carcinogenic and toxic lesion (Drabløs et al., 2004; Wedge et al., 1996; Wedge and Newlands, 1996). But MGMT, a suicide enzyme, causes re- covery of guanine by removing methyl addition. Unrepaired O6-MeG induces DNA mismatch repair (MMR) signal during DNA replication (Kyrtopoulos et al., 1997; Margison and Santiba´n˜ez-Koref, 2002). MMR recognizes the wrong mismatch timine in single strand and removes from strand. But O6-MeG remains in the other strand. This results in the addition of a thymine addition and cleavage cycle during replication. It ends the collapse of replication fork by causing DNA strand break (Mojas et al., 2007). Therefore, active MMR and low MGMT levels are impor- tant for a good response to TMZ.
MGMT promoteur contains numerous CpG dinucleotides (CpG islands) and epigenetic silencing of MGMT through the hyper- methylation of CpG is observed in % 30–60 of GBMs (Weller et al., 2010). This condition decreases the MGMT levels and increases cyto- toxicity of O6-alkylating agents, such as TMZ. During treatment in thevast majority of patients promoteur methylation status remain fix (Felsberg et al., 2011). This also suggests that the TMZ resistance is not relevant with alterations in methylation pattern of promoteur only. The weak therapeutic benefit of TMZ chemoradiotherapy, which shown in patients with promoteur methylated MGMT, raises the question whether these patients should receive TMZ or not (Brandes et al., 2008).

3. Hypoxia. Hypoxia inducible factor and its role in cancer metabolism
Hypoxia or low Oxygen tension is common property for all solid tumors. Tumor hypoxia is of clinical importance because it promotes both tumor progression and tumor resistance to radiation and chemo- therapy. HIF is a basal regulator that occurs against hypoxia and plays a
role in regulating a large number of genes necessary for the cell to adapt to hypoxia. HIF is a heterodimer consists one of two HIF-α subunits (HIF- 1α veya HIF-2α) and a HIF-1β. Tumor HIF-1α is an indicator of agressive disease and poor patient prognosis in cancer patients. As a result, HIF-1α is very important target for cancer treatment because of its role in tumor
survival and regulation of growth under hypoxic stress (Koh et al., 2009).
hydroxilated by Prolyl Hydroxilase Domain (PHD) protein. After, HIF-1α is directed to ubiquitination and proteosomal degradation by 26S pro- teosome (Maxwell et al., 1999). Under hypoxic conditions the hydrox- ylation decreases and HIF accumulates in cell (Kaelin and Ratcliffe, 2008). Hypoxia protect HIF-1α from PHD-dependent hydroxylation(Bruick and McKnight, 2001; Jaakkola et al., 2001). HIF-2α is expressed
in higher oxygen consentrations than HIF-1α (Holmquist et al., 2005; Wiesener et al., 1998).
In terms of therapeutic resistance, HIF-1 has been shown to suppress proapoptotic pathways that chemotherapeutic agents use to kill cancer cells, and same time has been observed to activate anti-apoptotic signal pathways (Erler et al., 2004; Chen et al., 2009; Peng et al., 2006).
Due to these properties of the HIF factor, different small molecules are developed, targeting HIF pathway (Semenza, 2003; Tan et al., 2005; Rapisarda et al., 2004). Most of them has indirect mechanisms, exhibit side effects, or have bad pharmacological properties (Belozerov and Van Meir, 2006, 2005). Many HIF-1α inhibitors have multiple targets. Inaddition, many of the currently available HIF-1α inhibitors target crucial
functions such as, cell signaling, DNA replication and cell division. At the same time some HIF-1α inhibitors exhibit their effect by inhibiting HIF-1α in different levels. For example, a guanylyl cyclase activator YC- 1 directs HIF-1α to degradation and inhibits its synthesis and intervenes HIF-1α/p300 interaction. As a result, suppresses HIF-1α by disrupting transcriptional activation (Li et al., 2008). Strikingly, numerous HIF-1α inhibitors affect in translational level. PX-478, another HIF-1α inhibitor, which in Phase 1 clinic trial inhibits HIF-1α by decreasing mainly the HIF-1α translation. Additionally, PX-478 can also decrease transcription and deubiquitination of HIF-1α (Welsh et al., 2003).
KC7F2 application has been reported to reduce HIF-1α levels and
inhibit activation of downstream HIF-1 target genes such as carbonic anhydrase IX, matrix metalloproteinase II, Enothelin I and Enolase I. KC7F2 inhibits cancer cell proliferation and its effect increases with hypoxia. Additionally, normal cells are less sensitive to this agent. KC7F2 is second generation HIF-1α inhibitor described by VanMaier group. The first proposed inhibitor 103D5R affects similarly by inhib- iting HIF-1α translation (Tan et al., 2005).

KC7F2 is cytotoxic against cancer cells under normoxic conditions and increases toxicity by rising HIF-1α levels under hypoxic conditions (Narita et al., 2009).
KC7F2 inhibits HIF-1α translation by decreasing phosphorylation of transcription suppressor eIF4E protein (4E-BP1) and ribosomal kinase S6K phosphorylation, and thus prevents translation initiation. HIF-1α is one of the critical proteins, expresses during hypoxia. This suggests that kc7f2 may be a mechanism that successfully performs the selective in- hibition of HIF-1α (Barnhart et al., 2008). KC7F2 is promising due to its higher selectivity for cancer cells compared to normal cells and increasing its cytotoxicity under hypoxic conditions. However, more studies are needed, targeting to evaluate the effect of drug from different aspects to make emerge the true nature and effective mechanism of KC7F2 (Koh et al., 2009).
4. Glucose metabolism

Carbohydrate metabolism is a critical and vital metabolic pathway provides energy for cancer cells by synthesizing ATP (Zhao et al., 2013). Thirty-eight molecules of Oxygen are being produced after complete catabolism of glucose in the presence of Oxygen. This process consists of 2 steps. These are glycolysis and mitochondrial steps. In the first step, glucose is metabolised and 2 molecules of ATP are synthesized. In the second step, mitochondrial phase, this process begins by entering the pyruvate into TCA cycle and after with the help of ETC it continuous till complete catabolism into CO2 and H2O. Finally, 36 molecules of ATP are synthesized (Warburg et al., 1927).

Glycolytic pathway consists of 12 basal components that 10 of them are enzymes. Each of these enzymes have different roles in glycolytic
pathway and converting cell metabolism into cancer metabolism. Additionally, a group of enzyme of this pathway are especially critical and have important functions by conversion of normal cell into cancer cell (Zhang et al., 2015a, 2015b).

In 1920’s Otto Warburg et al. observed that tumors intake more
amounts of glucose compared to environmental tissue. Because, these cells use more glucose molecule which to metabolize it until pyruvate. So the term aerobic glycolysis was appeared (Warburg, 1925). In this process unlike normal cells pyruvate is converted into lactic acid by LDHA enzyme. Consequently lactic acid accumulate in cells. These accumulated lactic acid molecules make cancer cell more acidic and trigger MCT transporters to throw these molecules out of the cell. Cancer cells prefer glycolysis to provide cells with energy and pyruvate is converted into lactic acid without entering TCA cycle. However, it is revealed that anaerobic respiration could maintain viability of tumor. Therefore, it was concluded that both oxygen and glucose should be eliminated to kill cancer cells by depriving them of energy (Warburg et al., 1927).

Later, in 1929, an English biochemist, Herbert Crabtree expanded Warburg‘s studies and examined heterogeneity of glycolysis in tumor types. He also confirmed Warburgs findings, however discovered that these process varied in different tumors (Crabtree, 1929).
As the findings from previous studies were not enough explanatory, later Warburg suggested that dysfunctional mitochondria is the main cause of aerobic glycolysis (Warburg, 1956). Warburg argued that this is the primary cause of cancer. This phenomenon was called as Warburg effect at the beginning of 1970s (Racker, 2018).

However, the role of the Warburg effect in cancer pathogenesis and tumor growth remained unclear till development of genetic and phar- macological studies (Fantin et al., 2006; Shim et al., 1998). Looking back at the original findings about tumor metabolism, the importance of targeting both aerobic glycolysis and mitochondrial metabolism was being understood (Flaveny et al., 2015; Sullivan et al., 2015). However, this remains controversial so far and the function of the Warburg effect on tumor growth is still unknown.
values were calculated by using CalcuSyn software (Biosoft).

5.3. Apoptosis analysis

To evaluate the apoptotic effects of KC7F2, TMZ and KC7F2 TMZ combination on the U87MG glioma cell line, 2.5×105 cells/well were incubated with the defined doses in 6-well plate and untreated cells were
used as control group. Annexin V-FITC (BD Pharminogen) kit and BD Accuri C6 Flow Cytometer (BD Biosciences) instrument were used ac- cording to kit protocol.

5.4. Quantitative protein analysis
To determine the changes of HIF-1α protein levels according to treatment of KC7F2, TMZ and KC7F2 + TMZ combination on the U87MG cells, Human HIF-1α ELISA Kit (FineTest. Cat. no EH0551) was
used in the study. Protein samples which were isolated from the treated and the untreated control groups were applied into the 96-well plates coated with Anti-HIF-1α antibody due to kit protocol. Finally, plates were read at 450 nm absorbance in Microplate reader (Multiskan, Thermo Fisher Scientific).

5.5. Metabolic analysis

The Seahorse XFp Cell Energy Phenotype Test (Agilent) and Seahorse XFp Extracellular Current Analyzer (Agilent) were used to measure the energy metabolism of the cells by determining the mitochondrial respiration and glycolysis rate under baseline and drug induced-stressed
conditions. To carry out this analyze, U87MG cells were seeded into Seahorse miniplates 1.25×104 cells/ 200 µl (well) concentration. IC50 doses of KC7F2, TMZ and KC7F2 TMZ combination were applied three
wells of the miniplate and untreated wells were used as control. After 48 h incubation of the cells in a non-CO2 incubator, the medium which includes the IC50 doses of the drugs, were removed and Agilent Seahorse XF Base Medium and stress inducers Oligomycin and FCCP were added into the wells according to Seahorse XFp Assay protocol and the analyze

5. Material and method
5.1. Cytotoxicity analysis
To determine the IC50 values of the TMZ and KC7F drugs, spectro-
was performed via Seahorse XFp (Agilent).

5.6. Gene expression analysis
ExtracellularCurrent Analyzerphotometric WST1 cytotoxicity analysis was used. U87MG glioma cells were seeded into 96 well-plate as 3.2×103 cells/well with 100 ml total volume in each well and were incubated for 24 h. Following the incu- bation period, KC7F2 (Sigma) and TMZ (Sigma) were administered to the cells at the dose ranges of 1–30 μM and 100–500 μM, respectively. Non-substance treated cells were defined as a control. Three replicates were used for each drug, dose and control group. At the end of each incubation period of 24, 48, and 72 h, 10 μl WST1 solution (Roche) was added into the wells and quantitative measurement of formazan dye intensity which occurred by live cells and tetrazolium salts are carried out by using microplate reader (Multiskan FC, Thermo Fisher Scientific) at 450 nm absorbance and 620 nm reference wavelengths every 15 min. IC50 values of the agents were calculated by using GraphPad Prism v5.0 software.

5.2. Isobologram analysis

Isobologram analysis is a widely used for assessing the combined effects of two different drugs on cells. To perform this analysis, the cells were seeded into the 96-well plate as the same concentration as cyto- toxicity analysis. After the 24 h incubation period, different dose com- binations of TMZ and KC7F2 which were based on the IC50 doses of the agents, were administered to the cells for 24, 48 and 72 h. The analysis was performed spectrophotometrically by WST1 test in the same way as cytotoxicity analysis. ED50, ED75, ED90, and Combination Index (CI)
To determine the changes in the expression levels of cellular energy metabolism-related genes, the IC50 doses of TMZ, KC7F2 and their combination were treated to U87MG cells for 48 h. The untreated cells were used as control. Total RNA was isolated from the cells using the RNeasy Plus Mini Kit (Qiagen) and cDNA synthesis was realized using RT2 First Strand Kit (Qiagen) according to the kit protocol. Glucose Metabolism (PAHS-006Z) arrays carried primers of the cellular energy metabolism-related 84 genes which 13 of these exhibited significant changes (Tables 1–3), were performed via RT2 SYBR Green qPCR Mas-termix (Qiagen) and the LightCycler 480 Instrument II (Roche). Fold changes of the genes were calculated by 2-Δ Δ Ct method, the fold change values > ±2 and p < 0.05 were considered as significant.

6. Results
6.1. TMZ and KC7F2 have effects on U87MG cell viability

In order to determine the appropriate concentration of U87MG human glioblastoma cell model, firstly cell proliferation assay was car- ried out via WST1 analysis. The TMZ and KC7F2 active substances were prepared at 100–500 μM and 10–30 μM dilution ranges, respectively. Untreated cells were used as control group. The cytotoxic effect was determined by the IC50 value, which is the dose concentration which reduces the proliferation of the cells by 50%. The IC50 values for KC7F2 was determined as 22.99 μM at 24th hour, 19 μM at 48th hour and 21.55μM at 72th hour. And the IC50 values for TMZ was determined as 512.1 μM at 24th hour, 461 μM at 48th hour and 531.5 μM at 72th hour. As can be seen the lowest IC50 values of TMZ and KC7F2 were determined as 461 μM and 19 μM at 48th hour, respectively (Figs. 1a and 1b).

6.2. TMZ and KC7F2 combination shows additive effect on U87MG cells

To determine the combinational effects of the TMZ and KC7F2 on the U87MG cell line, isobologram analysis was performed. According to the results, the CI values of TMZ and KC7F2 were found as 0.746 and the doses were 208.71 μM for TMZ and 8.60 μM for KC7F2 and the effects were accepted as additive (Fig. 2).

6.3. TMZ and KC7F2 induce apoptosis of U87MG cells both individually and in combination
Apoptotic effects of the IC50 and combination doses of the drugs
Temozolomid h treatment.cytotoxicityassay results after 24, 48 and 72were evaluated by using Annexin V method and flow cytometer at the end of 48 h. According to the results, in control group the apoptotic cell ratio was found to be 5.2%. In TMZ, KC7F2 and combination group it was 11.9%, 55.6% and 25.5%. As can be seen, TMZ, KC7F2 and their combination induced apoptosis 2-, 11-, and 5-folds more, respectively, compared to control group in U87MG cells (Fig. 3a,b, c and d).

6.4. KC7F2 decreases HIF1α protein levels

To evaluate the effects of TMZ, KC7F2 and their combination on HIF1α protein expression, standard curve and equation of HIF1α ELISA were created via reading of protein amounts range of 10000 pg/ml —
0.156 ng/ml at 450 nm. The HIF1α protein levels were determined using
this equation. According to the results the amount of HIF-1α protein in control group, TMZ, KC7F2 and drug combination applied groups was found to be properly 13280,5 pg/ml, 12323,5 pg/ml, 8608 pg/ml and 6373,5 pg/ml (Figs. 4a and 4b).

6.5. The drugs have significant effects on U87MG cell metabolism

As the result of the analyses, it was found that the metabolism in the cells treated with KC7F2 and drug combination tended to change from glycolytic to aerobic, compared to control groups (Figs. 5a–5c). In the KC7F2 treated group, the level of extracellular acidification rate (ECAR) was decreased 4- and 5- folds compared to the control group in normal condition and after stress condition, respectively. At oxygen consump- tion rates (OCR), 350-folds increase in the treated cells was observed in both normal and stress conditions. Oxygen consumption rates (OCR) were showed 350-folds increase in both conditions compared to the control. In the mentioned group, the metabolic potential was calculated as 61.24% and 109.75% according to OCR and ECAR, respectively. Although there was no significant difference in the ECAR in the TMZ

KC7F2 cytotoxicity assay results after 24, 48 and 72 h treatment.
treated group, OCR was increased 2.5 folds after stress application. In this group, metabolic potential was found as 209.03% and 180.71% according to OCR and ECAR, respectively. While no significant differ- ence was observed in the OCR in the drug combination group, the ECAR were decreased 20- and 35-folds compared to the control group in normal condition and after stress condition, respectively. In combina- tion group, metabolic potential was found as 102.61% and 164.01% according to OCR and ECAR, respectively (Figs. 5a–5c).

6.6. The effects of TMZ, KC7F2 and their combinations on gene expression levels
The fold changes of the expression levels of the energy metabolism-
related genes in U87MG cells due to treatment of TMZ, KC7F2 and their combination were calculated using 2-(ΔΔCt) method compared to control
group on 48th hours (Table 1). Genes exhibited significant changes at least in one treatment group are listed in tables below. In the KC7F2 treated cells, ALDOC, G6PC, GYS1, PCK1, PDK1, PDK3, PDK4, PGK1,
PGK2, PGM3, PRPS1L1, PYGL and TPI1 genes were downregulated 11.55, 5.50, 4.03, 33.59, 4.06, 2.85, 5.13, 2.87, 27.67, 3.20, 13.74, 2.01,
2.75 folds compared to the control group, respectively (Table 1).
In the Temozolomide treated cells ALDOC, GPI, GYS1, PDK1, PDK3, PGK1, PYGL, SDHA, TPI1 genes were downregulated 29,6097, 2,1258, 6,9067, 4,6525, 3,2898, 4,0784, 2,5991, 2,425, 2,3587 folds compared
to the control group respectively (Table 2).
In the TMZ and KC7F2 combination treated cells ALDOC, ENO2, GPI, GYS1, GYS2, HK3, PDK1, PDK3, PDK4, PGK1, PGK2, PRPS1L1, PYGL,
TPI1 genes were downregulated 23,6538, 2,1199, 2,0763, 290,8235,
11,6641, 4,1814, 3,2355, 3,4919, 3,691, 13,306, 2,0335, 2,1199,
2,3038 folds compared to the control group respectively (Table 3).

7. Discussion

Glioma is one of the most frequent subtypes of primary cerebral tumor that includes low and high grade types according to their ma- lignancy levels (Strowd, Holdhoff, and Grossman 2014). High grade glioma has metastatic phenotype and the median survival for patients with this cancer is only 13 months (Friedmann-Morvinski, 2014). Although conventional therapy for glioma consists of surgery and chemotherapy, relapse often follows the treatment. These facts show that new studies are required to develop more efficient treatment methods which includes targeted-therapy approaches (Li et al., 2017).TMZ, is a small lipophilic conventional chemotherapy agent, has ability to pass blood–brain barrier (Simonetti et al., 2014). TMZ is considered as an important chemotherapeutic drug for glioma because of it causes inhibition of cell growth and induction of cell death by inducing base mismatch and DNA abruption (Okita et al., 2015). How- ever, the barrier which decreases the effectiveness of TMZ, is created by

Results of KC7F2 and Temozolomide active ingredient combination. Isobologram analysis.
Evaluation of the apoptotic effects of KC7F2, Temozolomide and their combinations on U87MG glioblastoma cell line by Annexin V method. (here lower left quadrants represent live cell ratio, lower right quadrants represent apoptotic cell ratio, upper left quadrants represent necrotic cell ratio and upper right quadrants represent late apoptotic cell ratio).the O-6-methylguanine-DNA methyltransferase (MGMT), compels cli- nicians to raise the therapy doses (Li et al., 2017). Concordantly, find- ings from our study give rise to think that the effective dose of TMZ is extremely high levels and it would probable more effective to focus on
the dose reduction instead of developing a new drug. In our study IC50 dose of TMZ was determined as 461 μM and in combination with KC7F2 the effective dose of TMZ was approximately halved (208.71 μM).

As energy metabolism plays important roles in survival of cells, ATP is a common factor for all cell death mechanisms which contain different
characteristic molecular signaling pathways (Vanlangenakker et al., 2008; Skulachev, 2006).Study of Zhou et al. which included HT29-OxR and HCT116-OxR chemoresistant colorectal cancer cells, shed light on the correlation between aerobic glycolysis, ATP levels and drug resistance. It is deter- mined that chemoresistant cells had higher HIF-1α, cellular ATP, and aerobic glycolysis activity levels than non-resistant ones and the study showed that insertion of the liposome encapsulated ATP into the che- moresistant cell decreased HIF-1α and HK2 expression levels. This studye(Sankar et al., 1999; Hirose et al., 2001; Tong et al., 2001). The results we have obtained in our study support the treatment of TMZ but also suggest a proposal to increase its effectiveness.The individual and combination effects of the TMZ and KC7F2 on the energy metabolism were evaluated by gene expression method. Aldolase (ALDO) gene family members are important enzymes of glycolysis metabolism pathway. ALDO converts fructose 1,6-biphosphate (F1,6BP), a six-carbon product, into two three-carbon product dihy-
droxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate

Reference wavelength curve of HIF-1α ELISA kit.
Evaluation of the effects of KC7F2, Temozolomide agents and their combinations on HIF-1α protein levels by ELISA method.verifies the protective role of ATP for cancer cells and suggest that over activity of the glucose pathway and HIF-1α which plays a central role in glycolysis, have a significant contribution to development of resistance to therapeutic agents like as TMZ which was used in our study (Zhou et al., 2012). In our study, we observed that the HIF-1α protein level was decreased in the KC7F2 and combination groups compared to the con- trol group. This reveals the role of HIF-1α suppression in resistance to therapeutic agents such as TMZ. In order to confirm this, metabolic analyses were performed in the first stage of the study by considering the role of HIF-1 on glucose metabolism and the metabolic profile of the cells. In control cells, we observed that the function of both glycolytic and mitochondrial metabolism was realized, but the glycolytic meta- bolism was more active and the cells tended to glycolytic respiration. Although TMZ showed an inconsiderable effect on the metabolic profiles of the cells, in KC7F2 and combination treated cells mitochondrial respiration took the place of the glycolytic respiration. Considering the central role of HIF-1α in cancer glycolysis, KC7F2, the potential HIF-1α inhibitor, may have been caused to the change of metabolic profile, in correspondence to our hypothesis. Another study which includes KC7F2
showed that HIF-1α protein levels decreased in parallel with KC7F2 application dose in cancer cells. In addition, despite the KC7F2 showed lower toxic effect on normal fibroblast cells under normoxic conditions, its toxicity increased with HIF-1α level under hypoxic conditions (Tan et al., 2005). This finding proves the selective effect of KC7F2 on cancer cells and it also explains the effectiveness of TMZ and KC7F2 combination.
Numerous studies have demonstrated the weakened response and apoptosis resistance of glioblastoma cells to TMZ therapy and these reveal the necessity of researches to increase the TMZ effectiveness(G3P) (Prentki and Madiraju, 2008). They have also non-glycolytic functions. ALDOC is abundantly found in central nervous system, cere- brum, hippocampus, and Schwann cells (Prentki and Madiraju, 2008; Mukai et al., 1986; Xu et al., 2017; Caspi et al., 2014). The correlation between ALDOC expression, cell signaling pathways and tumourigenesis suggests that ALDOC might be effective in tumor formation and devel- opment (Caspi et al., 2014). In our study, it was observed that ALDOC gene expression level decreased significantly in all 3 groups treated with active substance compared to control. This actually shows that TMZ and KC7F2 suppress ALDOC gene expression level individually and in combination. Enolases (ENO) are intracellular catabolic enzymes that catalyze reversible 2-phospho-D-glycerate to phosphoenolpyruvate transformation dually (Pancholi, 2001; Kim and Dang, 2005; Dang and Semenza, 1999). ENO2, also called as gamma- or neuron specific- ENO, is a glycolytic pathway enzyme predominantly expressed in neurons and neuroendocrine system cells. It participates to accelerated glycolysis which supports increased cell metabolic demand and proliferation of cancer cells. C-terminal end protects tumor cells from stress conditions and defends the cell against the effects of therapeutic agents. Addi- tionally, it encourages migration and invasion of tumor cells by actin remodeling. These findings suggest that the role of this well-known tumor marker, whose expression has been altered during the develop- ment and progression of various cancers, is pleiotropic and still needs to be identified (Vizin and Kos, 2015). Although no significant changes were observed in the KC7F2 and TMZ applied group, 2-fold decrease was observed in the combination applied group. An important checkpoint in glycolytic pathway is metabolism of the pyruvate to acetyl-CoA by pyruvate-dehydogenase (PDK) (Yeaman, 1989; Reed, 2001; Harris et al., 2002; Fries et al., 2003). Tissue-specific four PDK isoenzymes are defined in human that are named as PDK1, PDK2, PDK3 and PDK4 (Popov et al., 1997; Bowker-Kinley and Popov, 1999). PDK1, which is a hypoxia sensitive enzyme, causes a decrease in mitochondrial oxygen consumption and prevents accumulation of reactive oxygen species through reducing mitochondrial function into acetyl-CoA in hypoxic conditions (Kim et al., 2006; Papandreou et al., 2006). Moreover, Wig- field et al. (Wigfield et al., 2008) showed that PDK1 associated with poor prognosis in head and neck cancers (Wigfield et al., 2008). This co- incides to the data we have obtained: PDK1, PDK3 and PDK4 were shown to be 4,2 and 5 folds suppressed in KC7F2 applied group, PDK1 and PDK3 were shown to be 4 and 3 folds suppressed in TMZ applied group, PDK1, PDK3 and PDK4 were shown to be 4,3 and 3 folds sup- pressed in combination applied group. No significant changes were observed in PDK4 in TMZ applied group. PGK1 balances the glycolysis by carrying out the first enzymatic step of ATP synthesis which includes 1,3-diphosphoglycerate to 3-phosphoglycerate conversion. Otto War- burg suggested that PGK1 may be the focus of metabolic alterations of glycolysis in malign processes than other metabolic enzymes (Warburg et al., 1927). According to Schneider et al., PGK1 inhibition provides to overcome chemoresistance by increasing vulnerability of tumor cells (Schneider et al., 2015). In our study, PGK1 expression level decreased in all treatment groups, especially in the KC7F2 treated group, and this
result indicates the role of HIF-1α factor in cancer metabolism. Tri-osephosphate isomerase (TPI) is a critical glycolytic enzyme which catalyzes DHAP to G3P conversion (Schneider et al., 2015; Brown et al., 1985). TPioverexpressed in lung adenocarcinoma, squamous cell carcinoma and breast carcinoma (Chen et al., 2002). Gess et al. reported that TPI upregulation is correlated with HIF-1α levels both in vivo and in

Evaluation of the effect of KC7F2 on u-87 mg glioblastoma cell metabolism with Seahorse xfp analysis method. Blue represent control cells and red represent KC7F2 treated cells.vitro (Gess et al., 2004). It is suggested that TPI upregulation can be triggered by a HIF-dependent pathway and HIF-1α may be the major promoter of TPI expression (Chen et al., 2017). In our data, although the expression of TPI was more downregulated in KC7F2 treated group, it was observed that the suppressive effect of TMZ was less than the combination and KC7F2. Although glucose storage as glycogen is also increased in various cancer types, glycogen levels are significantly
decreased due to glycogenolysis in some malignancies (Favaro et al., 2012; Pelletier et al., 2012) (Seitz and Luganova, 1968; Wagner, 1947). The biological results which are caused by increased amount glycogen are not known. Glycogen synthesis is controlled by Glycogen synthase (GYS) enzymes which include GYS1 and GYS2 subtypes. In contrast to GYS1, which is expressed at lower levels, GYS2 has great importance for glycogen synthesis in liver (Browner et al., 1989; Nuttall et al., 1994). Upregulated GYS1 and GYS2 were correlated with poor prognosis in acute myeloid leukemia, renal cancer, and lung adenocarcinoma (Bha- not et al., 2015). Considering that GYS1 is a glycogenolysis enzyme
induced by HIF-1α, combination therapy increased GYS1 expression
level by 1,9 folds more than KC7F2 treated group was observed in our study. Phosphoribosyl pyrophosphate synthetase 1 (PRPS1) which has

PRS1, PRS2 and PRPS1L1 isomers, catalysis the first step of phosphor- ibosyl pyrophosphate (PRP) synthesis from adenosine triphosphate (ATP) and ribose-5-phosphate (R5P) (Kornberg et al., 1955; Liu et al., 2013). The genes are highly conserved between chordates and they are expressed in different tissues like as brain suprarenal glands, lung and spleen (Taira et al., 1989; Yan et al., 2012). Qiu et al. reported that PRPS1 was upregulated both mRNA and protein levels in colorectal cancer (CRC) tissues and PRPS1 inhibition suppressed the colony for- mation and cell proliferation of CRC cells (Qiu et al., 2015). In a study of Chen Li et al. PRPS1 protein level was found increased in CD133 gli- oma cells (Li et al., 2016). This is supposed the main cause of encour- aging of de novo nucleotide biosynthesis to provide sufficient nucleotides in cancer cells (Bester et al., 2011; Tong et al., 2009). In fact, these studies support our data, and the effect of KC7F2 on this enzyme was firstly observed by us. Although no significant effect was observed in the TMZ-treated cell group, we found that significant levels of gene expression were suppressed in KC7F2 and in the combination group.This may be due to the effect of HIF-1α inhibition, which is perhaps the
main factor of metabolism. Glycogen synthase (GS) which elongates glycogen arms by creating α-1,4 glycoside bridges and Glycogen

Evaluation of the effect of Temozolomide on U-87MG glioblastoma cell metabolism with Seahorse xfp analysis method. Blue represent control cells and red represent TMZ (Temozolomide) treated cells.phosphorylase (GP) which converts these into glucose-6-phosphate and release glucose-1-phosphate, catalyzes the fundamental steps of degra- dation and synthesis respectively (Berg, 2002). Their activity can be modulated by posttranslational modification and allosteric effectors (ATP, AMP ve G6P) (Lawrence and Roach, 1997). Upregulation of PYGL which is a type of GP isomer, was reported in various cancer types including renal carcinoma, papillar renal cell carcinoma, seminoma and brain cancers compared to normal tissues (Tong et al., 2009; Berg, 2002). Its depletion which causes glycogen accumulation, is also asso- ciated with low proliferation and low survival (Favaro et al., 2012). These data suggest that PYGL may be the main enzyme to glycogenolysis
in HIF-1α-overexpressed cells. PYGL expression level was observed to be
decreased ~2 fold in all of three groups. Glucose-6-Phosphate Isomerase (GPI) is a basal glycolytic and glyconeogenic enzyme which catalyzes reversible isomerization between Glucose-6-phosphate (G6P) andfructose-6-phosphate (F6P). This step is found at the intersection of glycolysis and Pentose Phosphate Pathway (PPP) and plays a critical role in the fate of glucose metabolism. Metabolic glucose flux is directed by different PPP/glycolysis rate due to oxygen and glucose concentration in microenvironment and proliferation/differentiation status. Like many
glycolytic enzymes GPI expression is also induced by HIF-1α (Semenza,
2013) and increased in many cancers (Semenza, 2013; Pusapati et al., 2016). 2 different studies by Genentech reveals that pharmacological blockade of glycolysis through suppression GPI level reprograms the metabolism (Newgard et al., 1989; Winter et al., 2007). This study re- veals that most tumor cell lines analyzed had arrested their growth and this pharmacological approach directed cells to apoptosis. Studies in 8 different human cancer cell lines have revealed high GPI mRNA levels
varied between 2 and 10-fold. Both HIF-1α and VEGF is shown to induce
increased expression of GPI (Herling et al., 2011). In our study, the
Evaluation of the effect of combinations of Temozolomide and KC7F2 on U-87MG glioblastoma cell metabolism by Seahorse xfp analysis method. Blue represent control cells and red represent drug combination treated cellsincrease in GPI level as a result of TMZ and combination treatment revealed the effect of combination therapy on GPI. Considering GPI-HIF- 1α correlation our data proves the effect of combination treatment on metabolism. Glucose-6-phosphatase catalytic subunit (G6PC) takes part in the membrane of endoplasmic reticulum (Lei et al., 1993) and this enzyme plays physiological role on glycogenolysis. Its critical tumori- genic role on glioblastoma has been demonstrated in studies (Abbadi et al., 2014). In a study which includes sequence data of ovarian cancerpatients, increased G6PC levels were inversely correlated with survival. Following this data, suppressed G6PC is observed to decrease cell pro- liferation, viability, invasiveness and cell growth. G6PC silencing pro- vided also restoration of cell cycle proteins (Guo et al., 2015). The pharmaceutical inhibition of G6PC has similar effect to genetic silencing. This result suggests that the enzyme assumes two distinct roles in both glucose metabolism and cell cycle control, and these all make this enzyme a promising therapeutic target. In our study, G6PC expression level was shown to be decreased 5,5022 folds in the KC7F2 treated group. Despite 17,777 fold overexpression in TMZ-applied group and no any change observed in the combination group.

PCK1 and PCK2 are cytoplasmic and mitochondrial isoforms of Phosphoenolpyruvate Carboxykinase (PEPCK), the key enzyme of
protein level is higher in cancer cells than normal cells even in the normoxic state, and KC7F2 significantly affects cancer cell metabolism by suppressing this important factor. Moreover, KC7F2 effective dose was almost halved in combination group and this result suggests that combination effect is successful in this regard.

8. Conclusion

Resistance to the TMZ, which is used as a conventional chemother- apeutic therapeutic agent in glioblastoma cells, decreases the survival rate in patients and complicates the treatment. In addition, cancer cells which can use anaerobic glycolysis even under normoxic conditions make these cells more resistant. For this reason, in our study we inves-
tigated the effect of the HIF-1α suppression on DNA alkylating agent,
TMZ, sensitivity of glioblastoma cells. It was aimed possible reduction of therapeutic dose of the agent via combining KC2F7. It was also aimed to illuminate the results of inhibition of DNA alkylation and glucose metabolism, which are two distinct mechanisms on glioblastoma cells. As a result of the study, it was evaluated that the metabolism- targeted approach improves the cytotoxic and apoptotic effects of TMZ. Furthermore, we suggest that this study will contribute to the standard glioblastoma treatment through regulating critical metabolic
pathway-related genes.

Funding

The authors received no financial support for the research, author- ship, and publication of this article.
CRediT authorship contribution statement
Zaka Abbaszade: Conceptualization, Methodology, Software, Writing - original draft, Formal analysis, Project administration. Bakiye Goker Bagca: Writing - review & editing. Cigir Biray Avci: Supervision.

Declaration of Competing Interest

The authors declare that they have no known competing financial

gluconeogenesis, which converts oxaloacetate to Phosphoenolpyruvate (PEP) (Leithner, 2015). PCK2 has a unique position in cell metabolism via using alternative carbon sources such as lactate in biosynthetic pathways. In her own study, Leithner observed that the expression of this enzyme in lung cancer cells was increased (Leithner et al., 2015). Studies suggest that PCK1 plays an important role in the adaptation of cancer cells to glucose deprivation. In our study, 33-fold decreased expression was observed in KC7F2 treated group. Despite KC7F2, in both of TMZ and combination groups PCK1 expression levels was increased properly 3 and 1.999 fold. Interestingly, the glyconeogenic enzyme FB1 is a negative regulator of glycolysis which acts as downstream of PEPCK, was reported to be silenced in Pancreatic cancer (Zhang et al., 2015a, 2015b) and inhibit tumor progression in renal cancer. Therefore, tumor specific differences are needed to clarify the role of PEPCKs in cancer cell metabolism (Leithner, 2015). Our results determined that FBP1 level was overexpressed in all of 3 treated groups.
Mojsilovic-Petrovic et al. (2007) determined that HIF-1α protein
level exhibited 2 folds of increase in stimulated cells compared to control astrocyte cells (Mojsilovic-Petrovic et al., 2007). This data supports the ELISA results obtained from our study. Although increased levels of HIF- 1α protein in response to hypoxia in normal cells are physiological,
increased levels of HIF-1α protein are found in cancer cells even under
normoxic conditions. To test this case, KC7F2, TMZ and combination treated cell groups were evaluated by comparison with control cell group. As a result, it was observed that HIF-1α protein level was 2 folds decreased in KC7F2 treated group compared to control. Although this value decreased slightly in combination therapy, no significant change was observed in TMZ treated group. These data suggest that the HIF-1α
interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

I thank the referees for their suggestions in advance.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi. org/10.1016/j.gene.2021.145445.

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