Cytotoxic effect of TDZ on human cervical cancer cells

Accepted Manuscript Cytotoxic effect of TDZ on human cervical cancer cells

Gansukh Enkhtaivan, Doo Hwan Kim, Muthuraman Pandurangan PII: DOI: Reference:

S1011-1344(16)31162-9 doi: 10.1016/j.jphotobiol.2017.06.032 JPB 10893

To appear in:

Journal of Photochemistry & Photobiology, B: Biology

Received date: Revised date: Accepted date:

19 December 2016 23 May 2017 24 June 2017

Please cite this article as: Gansukh Enkhtaivan, Doo Hwan Kim, Muthuraman Pandurangan , Cytotoxic effect of TDZ on human cervical cancer cells, Journal of Photochemistry & Photobiology, B: Biology (2017), doi: 10.1016/ j.jphotobiol.2017.06.032

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ACCEPTED MANUSCRIPT Cytotoxic effect of TDZ on human cervical cancer cells Gansukh Enkhtaivan, Doo Hwan Kim, and Muthuraman Pandurangan* Department of Bio-resources and Food Science, Konkuk University, Seoul 143-701, South

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Korea.

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Running title: TDZ and HeLa cells

*Corresponding author: Muthuraman Pandurangan

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Assistant Professor

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Department of Bio-resources and Food Science

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Konkuk University, Seoul 143-701, South Korea Tel: +82-10-4733-7670 E-mail addresses: [email protected]

ACCEPTED MANUSCRIPT Abstract The present study investigates the anticancer activity of Thidiazuron (TDZ). Anticancer activity of TDZ was evaluated in cervical carcinoma cells (HeLa cells). Sulforhodamine-B (SRB) assay indicates that TDZ was about 100 times more toxic to the

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cancer cell than normal cells. TUNEL assay showed TDZ induced DNA damage in tumor

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cells. The loss of mitochondrial membrane potential (MMP) in cancer cells was observed

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following TDZ treatment. The Bax and bcl-2 gene expression ratio are highly responsible for the regulation of MMP balance, and these ratio was significantly altered following TDZ

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treatment. The p53 and caspase-3 expressions were increased in cancer cells following

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treatment. Caspase-3 activation is the key factor for apoptosis. Cytotoxicity of TDZ on HeLa cells was 100 times higher than normal kidney cell (MDCK cells). Moreover, the

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anticancer activity of TDZ was tested by DNA damage, mitochondrial dysfunction, some gene

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expression and caspase-3 inhibition in silico. TDZ detected has higher ability on early apoptosis of cancer cell through DNA damage. Additionally, cancer cellular MMP was

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significantly reduced under inoculation of TDZ. In silico assay confirmed that TDZ was able

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to bind with the active site of the capase-3 protein. Therefore, taking all these data together it is suggested that the TDZ may be a potential agent to act against cervical cancer cells.

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Keywords: Thiadiazol; Mitochondrial membrane potential; HeLa cells; Anticancer.

ACCEPTED MANUSCRIPT Introduction Modern days, cancer and cancer-related diseases increasing in developed countries due to the stress of life style and work (Black and Garbutt, 2002; Esch et al., 2002). In 2008, 12.7 million cancer patients were diagnosed, and about 7.9 million people died of cancer

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(Enkhtaivan et al., 2016). The majority of cancers, some 90-95% of cases, are due to

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environmental factors including tobacco (25-30%), diet and obesity (30-35%), infections (15-

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20%), radiation (up to 10%), lack of physical activity, stress and environmental pollutants (Kushi et al., 2012). The most common types of cancer are stomach, lung, breast, cervical,

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prostate, colorectal and stomach cancer. According to a global report in 2010, financial costs

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of cancer were estimated at $1.16 trillion US dollars per year (McGuire, 2016). Hence it needs to find more efficient and cheap drugs.

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Molecular docking on computer-aided drug development provides great help in the

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development of new generation anticancer drugs. Since, their discovery more than a decade ago, caspases, the proteases mediating the signaling pathway in programmed cell death (apoptosis),

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have been studied as an important therapeutic target (Jacobson et al., 1997). As one of the

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prototypical member in the class of executioner caspases, caspase-3 has been found to be activated in multiple signaling pathways in several different models of apoptosis (Porter and

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Janicke, 1999). It has been proven to be a useful target for reducing the amount of cellular and tissue damage in cell culture and animal models by specific inhibitors (Garcia-Calvo et al., 1998; Hotchkiss et al., 2000). Since its development, Thidiazuron (TDZ) has been extensively used as a pesticide, herbicide, and plant metabolite and plant growth regulator, in plant tissue culture (Mamaghani et al., 2009). The first intended use of TDZ was cotton defoliator in the category of plant growth

ACCEPTED MANUSCRIPT regulator (Lamas and Athayde, 1999; Sivanesan and Park, 2014; Yorgancilar and Erisen, 2011). TDZ has extensive use as herbicide owing to its high cytokine-like activity. A lower concentration of TDZ induces abscission leading to defoliation when applied to crops such as cotton. It has been used to promote fruit set and maintaining freshness. We investigated the

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cytotoxic effect of TDZ tested by SRB assay, DNA break, mitochondrial membrane potential

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loss and some apoptotic-related gene expression. Molecular docking simulation study was

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targeted on caspase-3. MATERIALS AND METHODS

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Reagents and Cell Culture

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TDZ (C9H8N4OS, 220.25 g/mol) was obtained from Sigma-Aldrich Co., Ltd (SigmaAldrich, South Korea). Madin-Darby Canine Kidney (MDCK cells) and Human cervical

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carcinoma cells (HeLa cells) were purchased from American Type Tissue Culture penicillin and

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Collection (ATCC, USA). Dulbecco’s Modified Eagle’s Medium,

streptomycin and fetal bovine serum (FBS) were purchased from Welgene Inc.,

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(Gyeongsan City, South Korea).

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Cytotoxicity and Anticancer assay

The cytotoxicity assay was according to Muthuraman et al., (2016). MDCK and HeLa

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cells (2 × 105/ml) were seeded in 96 well plates and maintained for 24 h under standard conditions (37 °C, and 5% of O2). Then, the medium was removed and washed with phosphate buffered solution (PBS). TDZ was treated in different concentrations as 0.4-454 µM in triplicate onto wells after new media were added to the wells. After 48 h of incubation with TDZ, the medium was removed and washed twice with PBS. Washed plates were fixed with 70% of acetone for 1 h at−4 °C. After fixing process, SRB assay was performed for cytotoxicity

ACCEPTED MANUSCRIPT and anticancer activity (Muthuraman et al., 2016c). Briefly, 100 μL of SRB (SulforhodamineB) solution (0.4% w/v in 1% acetic acid) and incubated at room temperature in a shaker. After removal of SRB solution, plates were washed 5 times with 1% of acetic acid and then dried. SRB stained 96 well plates were observed for the morphology of cells using reflected light

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540 nm, and inhibitory concentration of 50% (IC50) was calculated.

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microscope (20x) and added 10 mM of Tris-base for overnight. The absorbance was read at

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RNA isolation and Quantitative real-time PCR

Total cellular RNA was isolated from cells using TRIZOL reagent following the

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manufacturer's protocols (Invitrogen, USA). The isolated total RNA was reverse transcribed

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using c-DNA Synthesis Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions (Gansukh et al., 2016; Muthuraman, 2014). Quantitative real-time PCR (qPCR) was

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performed using specific primers (Table 1), and expressions of mRNA were normalized by

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glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. The amplification was carried out using SYBR Green Master Mix (Reaction volume: 25 µl, 25 cycles) (Bioneer, USA).

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Relative gene expression was calculated according to a 2−ΔΔCT method (Muthuraman et al., 2014).

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TUNEL assay

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)

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assay was carried out according to the kit method (Click-iT TUNEL Alexa Fluor 647, Life Technologies, USA). Briefly, 1 × 105 cells/ml was seeded onto the glass bottom confocal cell culture dish (SPL Life Sciences Co., Ltd. USA). After 24 h of adherence, cells were washed and treated with or without TDZ concentrations as 4.5 µM and 45.4 µM for 24 h. Cells were fixed (4% formaldehyde, 20 min) and then permeabilized (0.25% Triton X-100, 25 min). ClickiT and TdT reactions were carried out according to kit instruction (Muthuraman et al.,

ACCEPTED MANUSCRIPT 2016). TUNEL image was taken by Olympus FLUOVIEW FV1200 confocal microscope (40x) (Olympus Corporation, Tokyo, Japan). Mitochondrial Membrane Potential (MMP) assay MDCK and HeLa cells were treated with or without TDZ (4.5 1 µM and 45.4 µM) for

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24 h. At the end of treatment, cells were fixed with 4% of paraformaldehyde and

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permeabilized with 0.25% of Triton X-100. After fixation, cells were stained with MitoRed

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(Santa Cruz Biotechnology, South Korea) for 30 min and Hoechst 333254 (Santa Cruz Biotechnology, South Korea) for 20 min. At the end of staining, cells were washed thrice with

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PBS, and visualized by AxioVert200 inverted fluorescent microscopy (Carl Zeiss, Germany)

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with red/blue filters and recorded by digital imaging AxioVision software (scale bar represents 20 µm, Carl Zeiss, Germany) (Muthuraman et al., 2016).

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Molecular Docking Study

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In this study, well-known cancer-related protein caspase-3 used for molecular docking carried by AutoDock Vina (version 1.1.2) program (Trott and Olson, 2010; Muthuraman et

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al., 2016). The crystal structure of caspase-3 (PDB Code: 1RE1) downloaded from Protein Data

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Base (PDB, www.rcsb.org). The water molecules were removed from the crystal structure of caspase-3, and polar hydrogens were added using AutoDock Tools (version 1.5.6). Moreover,

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United partial atom charges of Kollman were assigned. For the docking simulation, grid box was integrated as 40(x) × 54(y) × 48(z) and centered at -41.3(x) × 92.9(y) × 21.9(z) for a pocket of the active site of caspase-3 (Muthuraman et al., 2016b). The best 9 poses and highest binding activities were calculated by the Root Mean Square Deviation between interactions of the atoms and clusters of the structures performed by AutoDock Vina. The docking result was visualized by PyMol Molecular Graphing System.

ACCEPTED MANUSCRIPT RESULT AND DISCUSSION Modern day, a number of cancer rate has been tremendously increasing due to environmental and social pollution. Till today, several important targets have been discovered for anticancer drug development. The recent statistic, 8.2 million deaths (14.6% of

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human mortality) caused by cancer in 2012 excluding skin cancer (Enkhtaivan et al., 2016;

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Muthuraman et al., 2016a). Thus cancer-related scientific research and understanding to develop

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more competitive and efficient drug research being the most priority.

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Normal and cervical tumor cells were treated with different concentrations of TDZ (0.4-454 µM). TDZ found to be very effective against cancer cells, and it inhibited cell growth up to 90%

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at maximum concentration (454 µM) used in this study. Moreover, cancer cell morphology

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was highly altered with TDZ even lower concentration of 4.5 µM. On another hand, TDZ showed no effect on normal MDCK cells up to 45.4 µM of TDZ, and even cellular morphology

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was not changed (Figure 1.A). However, it significantly inhibited MDCK cell growth at 454 µM

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of TDZ. Therefore, 454 µM of TDZ could be toxic to normal cell growth and hence, it was

(Figure 1).

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excluded for the further investigations. The results were appeared in a dose-dependent manner

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TUNEL staining, a TdT-mediated dUTP-biotin nick end labeling is a widely used method for in-vitro detection of DNA damage (Gavrieli et al., 1992; Huerta et al., 2007; Ning et al., 2002). TUNEL assay can also be used to detect cell damage due to necrosis, and cells are undergoing DNA repair. It has also been applied to measure the number of cleaved DNA ends caused by DNA damage in cultured cancer cells. The result obtained from TUNEL assay for the HeLa cell was shown in figure 2. There was no apoptosis at 0.4 µM of TDZ concentration. However, the apoptotic cell number was tremendously increased at 4.5 and 45.4 µM of TDZ. A

ACCEPTED MANUSCRIPT significant DNA damage was observed at the higher concentration of TDZ treated cervical carcinoma cells (Figure 2). In known scientific paradigm, one of the curious factors to break DNA in a cancer cell is a loss of mitochondrial function potential. MMP is one of the parameters for the mitochondrial function, is generated by

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mitochondrial electron transport chain that creates an electrochemical gradient by a series of redox reactions (Liu et al., 2002). This mitochondrial functional difference between normal and

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cancer cells can provide a significant biochemical basis for the development of cancer drugs

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targeting the mitochondria in the cancer cells (Wen et al., 2013). Alternatively, drugs which can induce the release of apoptotic factors from the cancer cell mitochondria can prove useful in

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causing the death of cancer (Brown and Borutaite, 2012).

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In this study, Mitochondrial and nuclear morphology were assessed by the Mito Tracker Red

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and Hoechst, respectively. The extended lace-like network of normal mitochondria was found in control cells. TDZ treatment (4.5 and 45.4 µM) significantly altered the mitochondrial

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morphology of cervical carcinoma cell. Mitochondria were condensed clump structure in TDZ

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treated cancer cells (Figure 3). When cells lost their mitochondrial membrane potential, it produces large amount of cytochrome-C, and that cytochrome-C induces further apoptotic

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pathways.

To further confirm apoptosis, we measured apoptotic marker gene expression by qPCR. HeLa cells were exposed to different concentration of TDZ (0.4-454 µM) for 24 h showed significant changes in the mRNA expression of p53, Bax, bcl-3 and caspase-3. The mRNA expression level of p53, Bax and caspase-3 were significantly up-regulated in TDZ treated cancer cells compared with controls. The mRNA expression of p53 was increased 1.1, 1.3, 1.7 and 1.8 fold following 0.4, 4.5, 45.4 and 454 µM of TDZ respectively. The mRNA expression of

ACCEPTED MANUSCRIPT Bax was increased 1.2, 1.5, 1.82 and 2.1 fold following 0.4, 4.5, 45.4 and 454 µM of TDZ respectively. The mRNA expression of caspase-3 was increased 1.2, 1.4, 1.9 and 2.1 fold following 0.4, 4.5, 45.4 and 454 µM of TDZ respectively. Interestingly, the mRNA expression of bcl-2 was reduced 1, 0.9, 0.7 and 0.7 following 0.4, 4.5, 45.4 and 454 µM of TDZ

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respectively. In background hypothesis of science, bax increases the loss of MMP, but that bax

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should be balanced by the level of bcl-2. If bcl-2 reduction occurred, that bax would be increased.

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Currently, in silico simulations study is routinely used in modern drug design and

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discovery programs to understand the gene pathway network as well as drug receptor interaction (Choudhury et al., 2014; Cully, 2015). It is evident from the previous literature that these

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computational approaches can intensely support, and help the design of novel, more potent

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inhibitors by deciphering the mechanism of drug-receptor interaction (Meshram et al., 2012). Induction of apoptosis through activation of caspase-3 makes it a promising target for

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designing anticancer drugs (Bhunia et al., 2015).

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In silico, molecular docking simulation was performed to investigate the possible binding

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modes of TDZ against an active site of the caspase-3 using AutoDock Vina program (Trott and Olson, 2010). AutoDock docking program is a public, academic standard in molecular docking

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studies (Adeniyi and Ajibade, 2013; Cagmat et al., 2012). The result suggested that the TDZ was able to bind to drug binding pocket of caspase-3. The binding site was located in the inner middle part of binding pocket (1RE1) (Figure 5). The best glide energy was recorded as -5.5 kcal/mol, suggesting the high binding affinity of TDZ to caspase-3 (Table 2). Conclusion Cytotoxicity of TDZ on HeLa cells was 100 times higher than MDCK cells. TDZ was found induce early apoptosis of cancer cell through DNA damage. MMP was significantly

ACCEPTED MANUSCRIPT reduced under inoculation of TDZ. Caspase-3 and p53 expressions were notably increased following treatment of TDZ. Moreover, in silico result showed that TDZ was able to bind with the active site of caspase-3. Taking all these results together, it is suggested that TDZ could be a potential therapeutic agent for the treatment of cervical carcinoma.

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Acknowledgements

This work was supported by the KU Research Professor Program of Konkuk University,

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Seoul, South Korea.

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Conflicts of interest: Author declares that they have no conflict of interest.

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ACCEPTED MANUSCRIPT Figure legends Figure 1: The effects of TDZ on MDCK and HeLa cells. The different concentrations (0.45454.0 µM) of TDZ were treated to the MDCK and HeLa cells. After 48 h of incubation, SRB assay was performed for microscopic observation (A) and cell viability

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determination on HeLa (B) and MDCK cells(C). Scale bar represents 100 µm.

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Figure 2: TDZ induced DNA damage on HeLa cells detected by TUNEL assay. Scale bar represents 50 µm.

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Figure 3: TDZ induces mitochondrial membrane potential in HeLa cells. (A) Fluorescent

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microscope images of TDZ treated HeLa cells after staining with MitoRed and Hoechst 333254. HeLa cells were treated with or without TDZ concentrations as 4.5 µM and 45.4

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µM for 24 h. Cells were visualized under fluorescent microscopy. (B) The relative

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fluorescent intensity of MitoRed of Hela cells treated with TDZ in different concentration

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(0.4 - 454 µM observed by fluorescent spectrophotometer. Scale bar is 20 µm Figure 4: Cancer-related gene expression induced by TDZ. HeLa cell was treated with different

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concentrations (0.4 - 454 µM) of TDZ for 48 h.

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Figure 5: Structure of contacting residues and surface depicting of caspase-3 (PDB Code: 1RE1) with TDZ.

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ACCEPTED MANUSCRIPT Table 1: List of primers used for the qPCR experiment

p53

Bax

Sequence

Bases

5′-TAACAGTTCCTGCATGGGCGGC-3′

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5′-AGGACAGGCACAAACACGCACC-3′

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5′-TGGAGCTGCAGAGGATGATTG-3′

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5′-GAAGTTGCCGTCAGAAAACATG-3′

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Type

5′-GAACCGGCACCTGCACAC-3′

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5′-CATGCTGGGGCCGTACAG-3

Bcl-2

5′-TTAGTGATAAAAATAGAGTTCTTTTGTGAG-3′

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5′-CTTCACCACCATGGAGAAGGCTG-3′

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5′-GACCACAGTCCATGCCATCACTG-3′

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GAPDH

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5′-TTAATAAAGGTATCCATGGAGAACACT-3′

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Caspase-3

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ACCEPTED MANUSCRIPT Table 2. Binding energies of the 2,4-D on Caspase-3 along with their Root Mean Square Distance value calculated by AutoDock Vina program. Distance from best mode RMSD lower bound.

RMSD upper bound.

-5.5

0

0

2

-5.3

12.907

16.05

3

-5.2

12.169

14.525

4

-5

2.752

6.764

5

-4.9

5.637

1.0228

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-4.8

8.29

11.044

7

-4.6

3.905

5.285

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-4.6

2.49

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-4.4

14.316

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mode

Affinity (kcal/mol)

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15.779

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Table 2: Binding energies of the 2,4-D on caspase-3 along with their Root Mean Square Distance value calculated by AutoDock Vina program. Distance from best mode

Best

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Affinity (kcal/mol) RMSD upper bound. 0

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RMSD lower bound. -5.5

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2

-5.3

12.907

3

-5.2

12.169

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-5

2.752

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-4.9

5.637

1.0228

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-4.8

8.29

11.044

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-4.6

5.285

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-4.6

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16.05

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14.525 6.764

2.49

3.534

14.316

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HIGHLIGHTS

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TDZ reduced cancer cell viability TDZ reduced MMP TDZ altered mitochondrial morphology TDZ altered apoptotic gene expression TDZ bind with active site of caspase-3

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    