Synergistic Inhibitory Effect of Maleic Anhydride Derivatives and Quercetin on Cervical Cancer Cells Mediated by Attenuating Cell Survival through Modification of the GSH System

doi:10.21203/rs.3.rs-690986/v1

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Synergistic Inhibitory Effect of Maleic Anhydride Derivatives and Quercetin on Cervical Cancer Cells Mediated by Attenuating Cell Survival through Modification of the GSH System

https://doi.org/10.21203/rs.3.rs-690986/v1

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Oxidative stress is considered apromotive factor in cervical cancer progression. Agents that decrease cellular GSH levels have been reported to modulate the proliferation rate of cancer cells. To explore a new therapeutic candidate for cervical cancer, we evaluated the effects of combined administration of a maleic anhydride derivative and quercetin on a human cervical cancer-derived (HeLa) cell line and human epithelial cell lines (HaCaT, THLE-3 and cultured primary cells). The combinatorial treatment sensitizes tumor cells through significant decreases in ROS and GSH levels, inducing apoptotic cell death, which was probably induced by disruption of cellular redox homeostasis. In addition, S-phase cell cycle arrest, decreased cell migration and clear evidence of changes in cytoskeletal actin and core morphology were observed in HeLa cells at 24 and 48 h after treatment. In an in vivo model, we observed an increase in the survival time of animals given the combination treatment, as well as tumor inhibition. Therefore, current data suggest that this alteration in the cellular redox state mediated through coadministration of established agentsmay be a promising chemotherapeutic approach.

Oncology

Toxicology

Clinical Pharmacology

apoptosis

oxidative stress

cell cycle arrest

migratory inhibition

quercetin

maleic anhydride derivatives.

Despite the emergence of novel targeted agents and the use of various therapeutic combinations, no curative treatment options are available for patients with advanced cancer. The magnitude of this problem demands the need for new therapeutic agents. Although there is effective screening for cervical cancer, this disease remains a healthcare problem in developing countries where effective screening programs are limited [1].

Under physiological conditions, cells have an adequate antioxidant defense system capable of neutralizing reactive oxygen species (ROS) [2], with glutathione being one of the most important components. In cells, glutathione is mainly in its reduced state (GSH) and, to a much lesser extent, in its oxidized state (GSSG). This is because the enzyme that "reduces" the tripeptide from its oxidized form, glutathione reductase, is constitutively active and inducible in situations of oxidative stress. In fact, the GSH/GSSG ratio within cells is often used as an "indicator" of the oxidative state of a cell and cellular toxicity. GSH is essential in the elimination and detoxification of carcinogens and affects cell survival. However, it also confers resistance to a series of chemotherapeutic drugs, and cellular and molecular processes that involve the bioavailability of glutathione determine tumor growth patterns, evidencing the participation of GSH as a determinant and critical growth factor in neoplastic cells [3]. Low levels of GSH trigger mitochondrial apoptosis, necrosis, and autophagy [4–5]. Agents that decrease cellular GSH levels have been reported to modulate the rate of cell proliferation and inhibit tumor growth [6–7].

Derivatives of maleic anhydride inhibit cell viability and induce apoptosis, probably through decreasing mitochondrial GSH levels [8, 9]. Through in silico studies, it has been proposed that 3,5-dimaleamylbenzoic acid (C1) and 3,5-dimaleimylbenzoic acid (C2), both derivatives of maleic anhydride, are highly selective for the SH group of GSH through conjugation reactions towards the 1,2-addition carbonyl group and have relatively high selectivity towards the olefinic carbons by 1,4-Michael-type addition [10, 11]. In the search for new alternative treatments, it is proposed that for cancer cell-targeted treatment, pharmacological agents that have pro-oxidant effects mediated by increasing the generation of ROS at the cytosolic level or by abrogating the antioxidant defense system in tumor cells should be considered. The main anticancer effect is achieved by promoting an oxidative stress process after the coadministration of maleic anhydride derivatives (C1 and C2).

Experimental evidence suggests that the flavonoid quercetin (Q) has several potential anticancer properties, including antioxidant, antiestrogenic, antiproliferative and anti-inflammatory properties [12, 13]. Q, as an anticancer treatment, has the ability to dissociate Bax and Bcl-xL and activate caspases [14]. Induction of apoptosis by Q has also been reported to occur through AMPK activation and p53-dependent cell death pathways in colon cancer cells [15]. Combined treatments with Q and other molecules, such as lanthanum, menadione, hydrogen peroxide and doxorubicin, demonstrate that Q is capable of enhancing cytotoxic effects on different human cancer cell lines (HeLa, T Jurkat, PC3, MCF7 and HepG2) [16, 17]. We show that Q treatment enhances the cytotoxic effects of C1 and C2 by sensitizing tumor cells through a significant increase in ROS production and a decrease in the GSH level, inducing cell death via apoptosis, which is probably mediated by disruption of cellular redox homeostasis. Our results support the hypothesis that the development of targeted agents that moderate GSH levels, such as Q, will enhance the ability of chemotherapeutic agents to induce cancer cell death.

2.1 Cell culture and treatment

The HeLa cancer cell line was provided by Dr. Jose E. Garrido, as a control, we used two human epithelial cell lines (HaCaT provided by Dr Enrique Pérez and THLE-3 obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and male rat Fischer-344 primary hepatocytes isolated following the method described by Berry and colleague with modifications [18]. We used an aqueous solution containing 0.2% dimethyl sulfoxide (DMSO, Sigma-Aldrich) as the vehicle for the compounds. Additionally, the optimal dose for quercetin (sc-203225A, Santa Cruz Biotechnology, CA, USA) was 50 mM, and the doses used for 3,5-dimaleamylbenzoic acid (C1 group) and 3,5-dimaleimylbenzoic acid (C2 group) were 0.01 mM; these compounds were synthesized by our research group [8]. These same doses were used for each of the compounds in the combined treatment, which consisted of initially administering Q and then administering C1 or C2 30 min later (group C1 + Q and group C2 + Q).

2.2 Cell viability assay

The effects of the treatments on the viability of HeLa cells were determined using a 3-(4,5)-dimethylthiazol(-2-y1)-3,5-diphenyl-tetrazolium bromide (MTT, Thermo Scientific, Rockford, USA) assay. The absorbance of the product was read at 590 nm using an ELISA microplate reader (iMark, Bio-RadCA, USA). Cell proliferation was calculated using the following formula: cell proliferation (%) = (mean absorbance of treated wells/mean absorbance of control wells) X 100%.

2.3 Cell cycle analysis

The effects of the compounds on the cell cycle distribution of HeLa cells were evaluated by flow cytometry analysis. The cells were incubated in a staining solution (176 µL of 1X PBS, 4 µL of 10 mg/mL RNase and 20 µL of 1 mg/µL PI; 200 µL per sample) for 40 min at 37°C. The cell cycle distribution was analyzed using a FACSCalibur system. Data were analyzed using ModFit LT 4.1 software.

2.4 Confocal fluorescence microscopy

After treatment, cells were fixed with 4% paraformaldehyde. The cells stained with the fluorescent nuclear dye Hoechst (H3570, Abcam, Cambridge, MA, USA), and a second fluorescent label, phalloidin (A12379, Thermo Fisher). The cells were mounted with Vectashield mounting medium (P36930, Thermo Fischer) on conventional slides. They were then observed under a confocal spectral Leica SP8 lightning microscope.

2.5 Migration assay

The cell migration assay was evaluated in vitro with modifications to the method described by Liang et al. [19]. The percent inhibition was determined using ImageJ image analysis software.

2.6 Western blot analysis

Nonspecific binding was blocked for 1 h at RT with slow shaking, followed by incubation with a specific primary antibody overnight at 4°C. The membranes were incubated with the peroxidase-conjugated rabbit IgG secondary antibody (65-6120, Thermo Fischer) for 1 h at RT. The membranes were developed using a LI-COR 3600 C-Digit Blot scanner (Biocompare, SSF, CA, USA). Bands were detected by a chemiluminescence reaction with the Luminol Kit (WBKLS0100, Millipore). The intensity of the bands was determined using ImageJ image analyzer software.

2.7 Detection of ROS levels

The level of ROS was analyzed using 2,7-dichlorofluorescin diacetate (DCFDA), which is a fluorescence probe sensitive to oxidation. The ROS assay was performed as described previously [20]. The fluorescence of DCFDA was measured using a Fluoroskan Ascent (Thermo Scientific) fluorometer at a ʎex of 480 nm and ʎem of 515 nm. The data were analyzed using GraphPad Prism version 7.0 (GraphPad Software, La Jolla, CA, USA).

2.8 Detection of GSH and GSSG

The method performed is reported to measure both oxidized glutathione (GSSG) and its reduced form (GSH). The method takes advantage of the reaction of GSH with OPT at pH 8 and that of GSSG with OPT at pH 12; GSH can be complexed to N-ethylmaleimide (NEM) to avoid GSH interference with GSSG measurement [21]. Results were obtained using a fluorometer at a ʎex of 350 nm and ʎem of 420 nm. The data were analyzed using GraphPad Prism software version 7.0.

2.9 Detection of murine L5178-Y lymphoma

The experimental induction of this neoplasia in syngeneic male BALB/c mice was carried out by means of transplantation of tumor cells (L5178-Y) taken from an animal with established cancer. Syngeneic male BALB/c mice (6–8 weeks of age, weighing 26 to 28 g) were management in accordance with the regulations of the Institutional Committee for the Care and Use of Laboratory Animals (CICUAL). The cells L5178Y were maintained in the peritoneal cavity of mice by serial intraperitoneal transplantation of 3.2x10⁶ viable L5178-Y cells suspended in a 0.9% NaCl solution.

2.10 Assessment of survival time

Three days after cell inoculation, 8 groups of mice (n = 6) were created, the mice were weighed, and the indicated chemical compounds were administered intragastrically every 24 h for 10 days (see Fig. 5). We used an aqueous solution containing 0.5% carboxymethyl cellulose (CMC) as the vehicle for the compounds. The optimal dose of quercetin 2 mg/kg of weight, and the doses used for 3,5-dimaleamylbenzoic acid (C1 group) and 3,5-dimaleimylbenzoic acid (C2 group) were 6 mg/kg of weight. These same doses were used for each of the compounds in the combined treatment, which consisted of initially administering Q and then administering C1 or C2 (group C1 + Q and group C2 + Q). Cyclophosphamide was used as a positive control because it is one of the first-line alkylating agents used as an antineoplastic agent to treat this type of tumor.

2.11 Assessment of tumor volume

Total tumor fluid was collected with a syringe, the ascites tumor cell count was determined, and cell counting was performed with a Neubauer hemocytometer using the trypan blue dye exclusion method and the following equation: TV (mm3) = 4π (A/2)2 × (B/2), where (A) is the minor tumor axis and (B) is the major tumor axis.

2.12 Statistical analysis

All the data are expressed as the mean ± SEM for each analysis. Statistical analyses were performed by using the unifactorial ANOVA test and Tukey’s multiple comparison tests using GraphPad Prism software version 7.0. Values were considered to be significant when the p value was < 0.05.

3.1 The synergism of C1 or C2 with Q inhibits cell viability in HeLa cells but not in noncancerous cells.

A cervical cancer cell line (HeLa cells), noncancerous human epithelial cell lines (HaCaT cells and THLE-3 cells) and healthy rat primary hepatocyte cultures (PC-Rat) were exposed to independent and combined administration of C1, C2 and Q at increasing doses for 12, 24 and 48 h, and the cytotoxic effect was evaluated by an MTT assay. The cytotoxic effect was significant for most of the HeLa cell groups evaluated at 12 h posttreatment (Fig. 1a). As an independent treatment, C1 had the greatest cytotoxic effect, with a reduction of 55.4% observed at 48 h posttreatment (Fig. 1c). The synergistic effects produced by the administration of the pro-oxidant compounds (C1 and C2) before quercetin was the most effective approach (C1 + Q group and C2 + Q group), presenting a reduction of 33% in cell viability at 24 h. At 48 h, this reduction increased to 64% with respect to the group administered vehicle (DMSO) (Fig. 1b, c). Noncancerous HaCaT, THLE-3 and PC-Rat cells did not show cytotoxicity when C1 or C2 was administered before quercetin; however, quercetin alone reduced HaCaT and THLE-3 cell viability and PC-rat viability by 10% at 48 h posttreatment (Fig. 1d, e, f).

3.2 Morphological damage and inhibition of cell migration

HeLa cells were analyzed by nuclear staining with Hoechst H3570 (cyan blue) and F-actin staining with phalloidin A12379 (red) (n = 3). Figure 2a shows HeLa cells at 24 h posttreatment. After 24 h, separation of the extracellular matrix and nuclear condensation were observed in all the experimental groups. Other events, such as actin degradation and the presence of pyknotic nuclei, were observed mainly in the combinatorial treatment groups (C1 + Q group and C2 + Q group). The number of pyknotic nuclei (indicative of fragmented DNA) was statistically significant (p < 0.001) in most of the groups, and the highest numbers were observed in the C1 + Q group and C2 + Q group, which had an average of 58% at 48 h posttreatment (Fig. 2b).

Cell migration was performed to assess the ability of the proposed treatments to inhibit cell migration. HeLa cells were exposed to each of the treatments for 48 h after making a scratch in the monolayer of cells in each of the groups (n = 6). Treatment with Q, C1, or C2 decreased cell migration by 13%, 10%, and 12%, respectively. The combinatorial treatments produced significant decreases in cell migration, with a 43% decrease for the C1 + Q group and a 41% decrease for the C2 + Q group (Fig. 3a, b).

3.3 Validation of apoptosis by assessing activation of caspase 3

To determine the type and extent of cell death, we analyzed whether apoptosis induces death in cervical cancer cells given the proposed treatment. Western blotting was performed in quadruplicate to evaluate caspase 3 levels. As shown in Fig. 3c, procaspase-3 was found at 37 kDa and 21 kDa, and the fragmentation corresponding to its active form was significantly observed in all the experimental groups. The highest levels were observed in the groups given combination treatment, with an average of 30% (p < 0.001) more fragmentation in those groups than in the control groups.

3.4 Cell cycle arrest

The exposure of cells to C1, C2 or Q compared to exposure to vehicle (DMSO) showed a strong retention (p < 0.0001) of cells in the G0/G1-phase at 24 h after treatment, with values of 62.27%, 57.13% and 53.24%, respectively. The groups C1 + Q and C2 + Q showed S-phase cell cycle distributions with values of 48.77% and 66.82%, respectively. Additionally, 51.23% of the cells in the C1 + Q group and 33.18% of the cells in the C2 + Q group were in the G0/G1 phase, and we observed a total loss of cells in the G2/M phase for both groups. A significant decrease in the G2/M-phase distribution was observed for all treatments (p < 0.0001) (Fig. 4a, b).

3.5 Intracellular ROS content modulation

HeLa cells undergoing Q treatment exhibited significantly decreased ROS levels compared to the DMSO group (control group, DMSO was the vehicle for treatments). Treatments with C1 or C2 did not produce significant changes in ROS levels compared with DMSO (Fig. 4c). The groups C1 + Q and C2 + Q did not show changes in ROS levels with respect to the control group. HaCaT cells showed a total decrease in ROS levels after treatment with Q, and C1 treatment produced a decrease in ROS levels compared with DMSO. Surprisingly, the groups C2, C1 + Q and C2 + Q did not show changes in ROS levels in HaCaT cells (Fig. 4d).

3.6 Induction of oxidative stress after depletion of GSH

In tumors, Q treatment decreases GSH, GSSG and GSG + GSSG levels and increases the GSH/GSSG ratio (Fig. 4e). The C1 and C2 groups exhibited decreased GSH and GSG + GSSG levels and GSH/GSSG ratios, with the exception of the oxidized glutathione level, compared with the DMSO group. The C1 + Q and C2 + Q groups showed significant decreases in reduced glutathione levels, de novo synthesis of glutathione and the GSH/GSSG ratio compared with the DMSO group. Additionally, these groups did not show significant changes in GSSH levels compared to the DMSO group.

3.7 In vivo tumor Inhibition

Survival times were determined for a murine model, and the data showed that C1, C2 and cyclophosphamide (C+) treatments increased the survival time compared to vehicle (NaCl) treatment (Fig. 5e, f). The combinatorial treatments C1 + Q and C2 + Q produced an average increase in the lifespan of 30 days compared to vehicle treatment and of 18 days compared with cyclophosphamide treatment. The group of animals administered quercetin alone did not exhibit an increase in survival time compared with the vehicle group. The results are presented in Fig. 5g, which shows monitoring of body weight. The body weight of the animals treated with C+, Q, C1 or C2 increased significantly, with an average value of 7.5 g more than that of animals in the vehicle group on day 26. A significant reduction in tumor volume was observed with the combinatorial treatments C1 + Q and C2 + Q compared with the other treatments, with inhibition percentages of 69.28% and 73.69% compared to vehicle treatment (Fig. 5g and h).

The association of tumor resistance to chemotherapeutic agents with high levels of GSH and GSSG in murine models and humans has been confirmed [22]. In recent years, tumor growth has been shown to depend on the bioavailability of glutathione, demonstrating its role as a critical growth factor in neoplastic cells [23–25]. In the search for new alternative treatments, treating cancer cells with molecules with pro-oxidant or antioxidant properties that change the mechanisms altered by ROS or modify the antioxidant defense systems of tumor cells has been proposed. The reduced state of thiol functional groups in amino acids, tripeptides, and proteins is important for cell proliferation [26]. The GSH content in tumor cells is a defense against oxidative stress [27]. An increase in the GSH level is also seen as an adaptive response for the disposal of electrophilic metabolites, many of which reduce the level of cellular glutathione by reacting with the sulfhydryl groups of GSH by binding covalently and nonspecifically to these nucleophiles in cellular macromolecules with consequent toxic effects [28, 29]. In addition to the changes cited above, depletion of GSH can induce a shift in the redox state in cancer cells, eventually leading to an increased probability of inducing DNA fragmentation and apoptosis [30].

The present investigation shows that GSH depletion in HeLa cells by combinatorial treatment (C1 + Q or C2 + Q) greatly increases cytotoxicity, cell cycle arrest, DNA fragmentation, caspase 3 activation, cell migration inhibition and glutathione system modification. In addition, the volume of L5178-Y lymphoma tumors induced in BAL/C mice was reduced, thus prolonging mouse survival time. This selectivity allows them to react with GSH and decrease its concentration in cancer cells. The maximum effect was induced after exposure to maleic anhydride derivatives followed by quercetin treatment. The combinatorial treatment did not affect noncancerous cells.

In cancerous cells, it was possible to observe changes in the morphology and population size, changes that were not observed in normal cells. These modifications in cancer cells were associated with programmed cell death. The Hoechst and phalloidin staining test allowed us to validate that the changes observed were attributed to events characteristic of apoptosis, such as the presence of pyknotic nuclei and F-actin disintegration. Sundaram, M. et al.,[31] reported changes in nuclear morphology in HeLa cells after administering quercetin at 25 or 50 µM for 24 and 48 h. Other authors have reported that F-actin labeling showed that the actin cytoskeleton of L929 cells begins to break down 2 h after treatment with quercetin [32]. Cell migration is a coordinated process that involves dynamic changes in the actin cytoskeleton and its interaction with focal adhesions. Our findings showed a clear effect producing a decrease in cell migration, an effect that was more evident with the combinatorial treatment. The pharmaceutical industry has focused on various types of molecules capable of inhibiting cell migration; however, the vast majority of them are still under study [33]. These results suggest that the combinatorial treatment could participate in tumor suppression by inhibiting cell migration.

Apoptosis in HeLa cells exposed to the treatments was validated by the activation of caspase 3, since caspases are the proteases that mediate the main morphological changes of apoptosis and various cell remodeling processes [34]. Our results show a clear effect on caspase 3 activation and indicate that combined treatments are the most effective in inducing caspase 3. Different studies have reported that GSH depletion induces arrest at different stages of the cell cycle in cancer cells [35, 36]. Cells undergoing uncontrolled proliferation develop high levels of GSH; in progression from the G1 phase to the S phase, cells require an increase in the content of nuclear GSH, and compounds with anticarcinogenic effects deplete the content of both cytosolic and nuclear GSH, arresting the cell cycle. The exposure of HeLa cells to C1, C2 or Q produced cell retention in the G0/G1 phase at 24 h, and the C1 + Q and C2 + Q groups showed S-phase arrest. We observed a significant decrease in the proportion of cells in the G2/M phase in all cases, and the C1 + Q and C2 + Q groups exhibited 100% decreases in this fraction. Clemente-Soto and collaborators showed that quercetin can induce G2-phase arrest and apoptosis in human cervical cancer-derived cells; this result was unlike what we observed in the group treated with quercetin alone or even in the combinatorial groups, where this phase was totally affected [37]. In human ovarian cancer cells, it has been shown that ARHI-aplasia Ras homolog member I can induce S-phase arrest, blocking proliferation and apoptosis, as we detected in this work [38]. The results obtained from this trial and in the development of the work allow us to predict that the levels of cellular GSH play an important role in the control of the cell cycle of cancer cells. It is imperative to point out that the present work proposes an alternative approach of combining maleic acid derivatives with quercetin and that the relevance is that this combinatorial therapy specifically alters cancer cells but not normal cells.

Acknowledgments

Wethank Eunice Romo Medina, Sergio Hernández García, Leobardo Molina García, Alejandro Cruz Hernández, Ma. Del Carmen NamoradoTonix and Jaime Escobar Herrera for their technical support.

Author Contributions

CTG and VRVG designed and performed experiments, conceptualization, wrote and edited the manuscript. EJMC, HMR performed experiments and analyzed data; JGTF, VRVG, and SVT, supervision of study, project administration, funding acquisition.

Funding

This work was supported by a scholarship from CTG, which was granted by the Consejo Nacional de Ciencia y Tecnología (CONACyT); project 178558 of SVT, which was funded byCONACyT; and the project CONACYT 2014-2499and 290194 of VRVG; CONACyT 2015-270189 of Dr. RafaelBaltierrez-Hoyos

Availability of data

All the findings generated during the study are included in this article. Any additional information may be provided from the corresponding author on reasonable request.

Ethical approval

All protocols carried out in animal studies were approved by the Institutional ComitéInstitucional para elCuidado y Uso de Animales de Laboratorio (CICUAL) of the Unidad de Producción y Experimentación Animal de Laboratorio (UPEAL) - CINVESTAV, following all international and national guidelines.

Consent to participate

Not applicable.

Informed consent

Not applicable.

Code availability

Not applicable

Consent for publication / Conflict of interest

All authors are aware of and agree with the content and presentation of this work and its submission to the journal Investigational New Drugs. All authors declare that they have no conflict of interest.

CaC, cervical cancer; Q, quercetin; C1, 3,5-dimaleamylbenzoic acid; C2, 3,5-dimaleimylbenzoic acid;ROS, reactive oxygen species; GSH, reduced glutathione; GSSG, oxidized glutathione;DMSO, dimethyl sulfoxide; MTT, 3-(4,5)-dimethylthiazol(-2-y1)-3,5-diphenyl-tetrazolium bromide; DCFDA, 2´,7´-dichlorodihydrofluorescein diacetate.

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Synergistic Inhibitory Effect of Maleic Anhydride Derivatives and Quercetin on Cervical Cancer Cells Mediated by Attenuating Cell Survival through Modification of the GSH System

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Cell culture and treatment

2.2 Cell viability assay

2.3 Cell cycle analysis

2.4 Confocal fluorescence microscopy

2.5 Migration assay

2.6 Western blot analysis

2.7 Detection of ROS levels

2.8 Detection of GSH and GSSG

2.9 Detection of murine L5178-Y lymphoma

2.10 Assessment of survival time

2.11 Assessment of tumor volume

2.12 Statistical analysis

3. Results

3.1 The synergism of C1 or C2 with Q inhibits cell viability in HeLa cells but not in noncancerous cells.

3.2 Morphological damage and inhibition of cell migration

3.3 Validation of apoptosis by assessing activation of caspase 3

3.4 Cell cycle arrest

3.5 Intracellular ROS content modulation

3.6 Induction of oxidative stress after depletion of GSH

3.7 In vivo tumor Inhibition

Discussions

Declarations

Abbreviations

References

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